Ophthalmic compositions and methods of use therefor

ABSTRACT

The present invention encompasses ophthalmic compositions that may be used for various conditions of the eye, and particularly, conditions of the cornea. Also encompassed are methods that utilise these compositions and kits that include these compositions.

RELATED APPLICATION

This application claims the benefit of New Zealand provisional patentapplication number NZ 705727, filed 5 Mar. 2015, the contents of whichare hereby incorporated herein in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods useful forthe treatment and/or prevention of conditions of the eye. In particular,the disclosure relates to compositions and methods that can be used inaugmenting and regenerating the cornea, and in correcting refractiveerrors of the eye.

BACKGROUND OF THE INVENTION

It was previously believed that differentiated cells relinquished theirability to regress to an earlier state. However, this view has beenchallenged by the induction of pluripotent stem cells (cellreprogramming) and evidence showing that differentiated cells can switchto another phenotype (Takahashi & Yamanaka 2006; Wernig et al. 2007;Yamanaka & Blau 2010; Gurdon & Melton 2008; Peran et al. 2011). Inaddition, it is now believed that the microenvironment for cells, whichincludes the surrounding cells, extracellular matrix, and growth anddifferentiation factors, plays an important role in bringing about theredirection of cellular differentiation (Håkelien and Collas 2002). Withthis information, researchers have begun to develop therapeutics thatutilise cell reprogramming and stem cell technologies.

The cornea of the eye accounts for more than two-thirds of the eye'stotal refractive power (focusing power). Even small changes in cornealshape can have a dramatic effect on the clarity with which an image isbrought to focus on the retina. The stromal layer of the cornea (theclear front surface of the eye) comprises the majority of the cornealtissue and is composed of highly organised lamellae which are made up oftightly packed collagen fibrils, mostly of collagen types I and V(Marshall et al. 1993). The unique structure of the stromal layer as aresult of the uniform alignment of the collagen fibrils confers theproperties of toughness and transparency on the cornea (Funderburgh2000).

When stromal cells (the corneal keratocytes) are removed from the corneaand cultured in a monolayer they exhibit the morphologicalcharacteristics of fibroblasts and switch from a stellate shaped cell toa multinucleate, fusiform shaped cell (Funderburgh et al. 2001). Anothercommonly observed phenotype of keratocytes is the myofibroblast formthat is seen in the cornea after injury (Jester et al. 1987). Changes inexogenous growth factors and cytokines are thought to bring about thesephenotypic changes (Funderburgh et al. 2001).

TGF family of growth factors are known to be the most potent inducers ofchondrogenic (cartilage) differentiation (Heng, Cao, & Lee 2004;Johnstone et al. 1998; Menetrey et al. 2000). TGFβ1 stimulates thesynthesis of collagens and fibronectin by chick embryo fibroblasts(Ignotz and Massague 1986). For keratocytes, TGFβ1 and TGFβ2 are knownto cause ECM deposition associated with scarring, possibly due toconversion of keratocytes into the myofibroblast phenotype (Funderburgh,Mann, Funderburgh, Corpuz, & Roth 2001). In contrast, TGFβ3 has beenshown to induce corneal fibroblasts to produce ECM depositions made upof collagen type I without fibrosis or scarring (Karamichos, Hutcheon, &Zieske 2011). Certain non-proteinaceous chemical compounds such asdexamethasone (Johnstone, Hering, Caplan, Goldberg, & Yoo 1998),ascorbic acid (Farquharson, Berry, Barbara Mawer, Seawright, & Whitehead1998), and ethanol (Kulyk & Hoffman 1996) are also known to promotechondrogenic differentiation in vitro.

There are a number of conditions affecting the cornea, including variousdefects, injuries, diseases, and degenerative conditions. Myopia resultsfrom excessive curvature of the cornea so that light entering the eyefocuses in front of the retina. It is the most prevalent visionimpairment worldwide affecting the vision of 70 to 90% of people in someAsian countries and 30 to 40% in Europe and the United States (Frederick2002). In most cases, myopia first occurs in school-age children andprogresses until about the age of 20. It is also associated withincreased prevalence of macular degeneration, retinal detachment, andglaucoma in adulthood (Ebenstein & Pruitt 2006).

Myopia is most commonly corrected by the use of prescription eye glassesor contact lenses. However, these devices do not provide permanenttreatment for the condition, and they are unsuitable for use duringcertain activities. Contact lenses are also associated with ophthalmicinfections and more serious conditions, including corneal abrasions andulcers. In certain circumstances, refractive surgery or orthokeratologyis indicated for myopia. Still, these treatments provide only atemporarily correction for mild to moderate myopia; they are notpermanent treatments, and they are unsuitable for severe cases.

Keratoconus is an ecstatic corneal dystrophy associated with stromalthinning and disruption of the portion of the cornea known as Bowman'slayer. The progressive thinning of the corneal stroma typically occursover decades and results in the cornea developing a conical shape. Thisresults in an impairment of vision due to irregular astigmatism andmyopia. The pathogenesis of keratoconus is still unknown but has beenassociated with factors such as constant eye rubbing and contact lenswear (Krachmer, Feder, & Belin 1984; Sherwin & Brookes 2004). It canappear as early as puberty and continues to progress until the third orfourth decade of life.

The incidence of keratoconus has been estimated at approximately 1 in2000 in the general population worldwide (Rabinowitz 1998), with nopredilection for either gender. Since the onset of keratoconus istypically in early adulthood with continuation into prime earning andchild-rearing years, the loss of quality of life and the economic burdenof the treatment of keratoconus represent a significant public healthconcern. Keratoconus is a major indication for cornea transplantation inthe Western world, determined by researchers to constitute 28.8% ofcorneal transplantation in France (Legeais et al. 2001) and from 11.4%to 15.4% in the United States (Cosar et al. 2002; Dobbins et al. 2000).There is an unusually high prevalence of keratoconus in New Zealand,with a disproportionately high incidence in Pasifika and Maoripopulations (Patel et al. 2005; Patel & McGhee 2013). In New Zealand,approximately 50% of all corneal transplants performed are forkeratoconus (Edwards et al. 2002).

Despite several studies on keratoconus, the underlying biochemicalprocess remains poorly understood. The familial occurrence ofkeratoconus suggests that one of the aetiological factors is genetic(Ihalainen 1985). The condition has also been linked to certainbiochemical and biomechanical factors. For example, it has beendetermined that the corneal thinning of keratoconus is a result of theloss of extracellular matrix (ECM) components. However, this could bedue to their destruction, their defective formation, or a combination ofthese (Klintworth & Damms 1995; Klintworth 1999; Jhanji et al. 2011). Inthe corneal stroma, changes associated with keratoconus include adecrease in the number of lamellae and keratocytes (Ku, Niederer, Patel,Sherwin, & McGhee 2008; Sherwin & Brookes 2004), and changes inorganisation of the lamellae and distribution of collagen fibrillarymass (Meek et al. 2005).

It is thought that the degradation of the stromal layer might be due toaberrant proteolytic enzyme activity (Fukuchi, Yue, Sugar, & Lam 1994).Keratoconus corneas are known to have decreased levels of enzymeinhibitors and an increased level of degradative enzymes (Kenney & Brown2003). Biomechanical factors include thinning and decreased rigidity ofthe cornea due to oxidative damage caused by ultraviolet radiation andmechanical trauma (Kenney & Brown 2003). Biomechanical investigation ofkeratoconic corneas has revealed a decrease in elasticity and stiffness;however the reasons for this remain unknown (Edmund 1988). It has beensuggested that a reduction in collagen cross-links could be a cause(Wollensak & Buddecke 1990). Currently there is no satisfactory animalmodel for keratoconus and investigations have been largely limited to anex vivo setting.

Depending on the severity of the condition, attempts to slow progressionof keratoconus include the use of special spectacles and contact lens.In severe cases, corneal implants, intrastromal rings, or cornealtransplants are necessary (Jhanji, Sharma, & Vajpayee 2011). Penetratingkeratoplasty, a procedure in which the entire thickness of the cornea isremoved and replaced by donor corneal tissue, is the most commonly usedsurgical procedure used to treat advanced cases of keratoconus(Rabinowitz 1998). Keratoconus is the leading indication for cornealtransplantation surgery worldwide, with about 12-20% of those affectedby keratoconus requiring a corneal transplant (Pramanik, Musch, Sutphin,& Farjo 2006).

Early treatment options for keratoconus, such as customised gaspermeable lenses known as Rose K lenses, have been focussed on improvingvisual acuity. Newer treatments aim to slow the progression of thedisease. A treatment known as corneal collagen cross-linking (CXL) looksat increasing corneal rigidity and biomechanical stability. In thisprocedure, the epithelium is debrided, topical riboflavin drops areadministered, and the corneas are exposed to ultraviolet-A light at 370nm for approximately 30 minutes (Ashwin & McDonnell 2010; G. Wollensak,Spoerl, & Seiler 2003). It is believed that the UV-A light activates theriboflavin thereby producing reactive oxygen species that induce theformation of covalent bonds between the collagen molecules in thecorneal stroma (Spoerl, Huhle, & Seiler 1998; G. Wollensak et al. 2003).This procedure, however, is not recommended for the treatment of corneasthinner than 400 μm due to the possibility of endothelial cell damage.Although this treatment leads to a stiffer cornea, it does not addressthe problem of corneal thinning.

Therefore, there is an ongoing need for therapeutic compositions andmethods for addressing conditions of the eye, including conditionsaffecting the cornea. There is a particular need for therapies that arerelatively non-invasive and readily administered.

SUMMARY OF THE INVENTION

The inventors have developed compositions and methods for modulatingcorneal cells, to alter collagen expression and extracellular matrixformation in corneal tissue. These compositions and methods are usefulfor regenerating and/or augmenting the cornea, and thereby treatingand/or preventing various conditions of the cornea and refractive errorsof the eye.

In one aspect, the invention comprises a method of treating orpreventing a condition associated with a thinning or irregularity of acornea, comprising: contacting the cornea with a composition comprisinga TGFβ3 polypeptide or a variant or fragment thereof, and dexamethasoneor derivative thereof or related steroidal agent, thereby treating orpreventing the condition.

In various aspects:

The TGFβ3 polypeptide consists of the amino acid sequence of SEQ IDNO:1.

The dexamethasone is dexamethasone phosphate.

The composition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide.

The composition comprises 40 to 4000 ng/ml dexamethasone.

The composition is formulated as an eye drop.

The composition is formulated with gellan gum.

The composition is administered once daily or twice daily.

The composition is co-administered with one or more additional agentsfor the eye.

The one or more additional agents for the eye are selected from thegroup consisting of: anaesthetic agents, anti-inflammatory agents,anti-microbial agents, and lubricants.

The composition is administered in conjunction with use of a contactlens, corneal insert, corneal implant, or intrastromal ring.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted for moulding or holding corneal shape during and/or followingtreatment with the composition.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted to act as a carrier for the composition or as a compositioneluting device.

The composition is administered in conjunction with corneal collagencrosslinking.

The administration of the composition is prior to and/or subsequent tocrosslinking.

The condition is selected from the group consisting of: keratoconus,myopia, and astigmatism.

In an alternative aspect, the method comprises co-administration of acomposition comprising the TGFβ3 polypeptide or a variant or fragmentthereof, and a composition comprising the dexamethasone or derivativethereof or related steroidal agent.

In one further aspect, the invention comprises a method of treating orpreventing damage or injury of a cornea, comprising: contacting thecornea with a composition comprising a TGFβ3 polypeptide or a variant orfragment thereof, and dexamethasone or derivative thereof or relatedsteroidal agent, thereby treating or preventing the damage or injury ofthe cornea.

In various aspects:

The TGFβ3 polypeptide consists of the amino acid sequence of SEQ IDNO:1.

The dexamethasone is dexamethasone phosphate.

The composition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide.

The composition comprises 40 to 4000 ng/ml dexamethasone.

The composition is formulated as an eye drop.

The composition is formulated with gellan gum.

The composition is administered once daily or twice daily.

The composition is co-administered with one or more additional agentsfor the eye.

The one or more additional agents for the eye are selected from thegroup consisting of: anaesthetic agents, anti-inflammatory agents,anti-microbial agents, and lubricants.

The composition is administered in conjunction with use of a contactlens corneal insert, corneal implant, or intrastromal ring.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted for moulding or holding corneal shape during and/or followingtreatment with the composition.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted to act a carrier for the composition or as a compositioneluting device.

The damage or injury of the cornea is associated with one or more of: anabrasion, tear, ulcer, burn, puncture, and surgery.

In an alternative aspect, the method comprises co-administration of acomposition comprising the TGFβ3 polypeptide or a variant or fragmentthereof, and a composition comprising the dexamethasone or derivativethereof or related steroidal agent.

In yet a further aspect, the invention comprises a method of treating orpreventing a refractive error of the eye, comprising: contacting theyeye with a composition comprising a TGFβ3 polypeptide or a variant orfragment thereof, and dexamethasone or derivative thereof or relatedsteroidal agent, thereby treating or preventing the refractive error ofthe eye.

In various aspects:

The TGFβ3 polypeptide consists of the amino acid sequence of SEQ IDNO:1.

The dexamethasone is dexamethasone phosphate.

The composition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide.

The composition comprises 40 to 4000 ng/ml dexamethasone.

The composition is formulated as an eye drop.

The composition is formulated with gellan gum.

The composition is administered once daily or twice daily.

The composition is co-administered with one or more additional agentsfor the eye.

The one or more additional agents for the eye are selected from thegroup consisting of: anaesthetic agents, anti-inflammatory agents,anti-microbial agents, and lubricants.

The composition is administered for use in conjunction with a contactlens, corneal insert, corneal implant, or intrastromal ring.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted for moulding or holding corneal shape during and/or followingtreatment with the composition.

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted to act as a carrier for the composition or as a compositioneluting device.

The method is performed preceding or following refractive surgery.

The refractive error of the eye is associated with one or more of:myopia, hyperopia, astigmatism, and presbyopia.

In an alternative aspect, the method comprises co-administration of acomposition comprising the TGFβ3 polypeptide or a variant or fragmentthereof, and a composition comprising the dexamethasone or derivativethereof or related steroidal agent.

In still a further aspect, the invention encompasses a kit comprising:

a composition comprising a TGFβ3 polypeptide or a variant or fragmentthereof, and dexamethasone or derivative thereof or related steroidalagent; and

one or more contact lenses.

In various aspects:

The contact lens, corneal insert, corneal implant, or intrastromal ringis adapted for moulding or holding corneal shape during and/or followingtreatment with the composition.

The contact lens, corneal insert, corneal implant, or intrastromal ringact as a carrier for the composition or as a composition eluting device.

The TGFβ3 polypeptide consists of the amino acid sequence of SEQ IDNO:1.

The dexamethasone is dexamethasone phosphate.

The composition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide.

The composition comprises 40 to 4000 ng/ml dexamethasone.

The composition is formulated as an eye drop.

The composition is formulated with gellan gum.

The composition is formulated for administration once daily or twicedaily.

The composition is co-formulated with one or more additional agents forthe eye.

The kit includes one or more additional agents for the eye.

The one or more additional agents for the eye are selected from thegroup consisting of: anaesthetic agents, anti-inflammatory agents,anti-microbial agents, and lubricants.

The kit includes a contact lens solution.

The kit includes instructions for use.

The kit is used for the treatment or prevention of a refractive error ofthe eye.

The kit is used for the treatment or prevention of a corneal conditionselected from the group consisting of: keratoconus, myopia, hyperopia,astigmatism, presbyopia, and stromal dystrophies.

The kit is used for the treatment of a corneal condition selected fromthe group consisting of: an abrasion, tear, ulcer, burn, puncture,corneal melt, and surgical injury.

In an alternative aspect, the kit comprises as separate components acomposition comprising the TGFβ3 polypeptide or a variant or fragmentthereof, and a composition comprising the dexamethasone or derivativethereof or related steroidal agent.

In even a further aspect, the invention comprises a method of inducingcollagen type II expression in a keratocyte, comprising: contacting thekeratocyte with a composition comprising a TGFβ3 polypeptide or avariant or fragment thereof, and dexamethasone or derivative thereof orrelated steroidal agent, thereby inducing collagen type II expression inthe keratocyte.

In various aspects:

The TGFβ3 polypeptide consists of the amino acid sequence of SEQ IDNO:1.

The dexamethasone is dexamethasone phosphate.

The composition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide.

The composition comprises 40 to 4000 ng/ml dexamethasone.

The composition is formulated for administration via a contact lens, acorneal insert, a corneal implant, or an intrastromal ring.

The composition is formulated for administration as a solution, gel,cream, or emulsion.

The method is performed in vivo.

The method is performed ex vivo.

In an alternative aspect, the method comprises co-administration of acomposition comprising the TGFβ3 polypeptide or a variant or fragmentthereof, and a composition comprising the dexamethasone or derivativethereof or related steroidal agent.

The foregoing brief summary broadly describes the features and technicaladvantages of certain embodiments of the present invention. Furthertechnical advantages will be described in the detailed description ofthe invention and examples that follows.

Novel features that are believed to be characteristic of the inventionwill be better understood from the detailed description of the inventionwhen considered in connection with any accompanying figures andexamples. However, the figures and examples provided herein are intendedto help illustrate the invention or assist with developing anunderstanding of the invention, and are not intended to limit theinvention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Schematic showing myopia caused by an increased curvature ofthe cornea such that light entering the eye is not focussed onto theretina.

FIG. 1B: View of a normal (A) and keratoconic (B) cornea, respectively.(C): Scheimpflug image in severe keratoconus. Significant cornealthinning is appreciated in the central cornea.

FIG. 2: Organotypic slice culture set up.

FIG. 3: Growth factor eye drops instilled in the eye of adult maleWistar rat.

FIG. 4: Phoenix Micron IV in vivo eye imaging system set up specific forimaging rat eyes.

FIG. 5: Schematic of (A): a nanoindenter system, (B): a typicalload-displacement curve obtained during the indentation process which isused to calculate corneal elasticity and hysteresis. P_(max)=maximumload applied; h_(max)=penetration depth; h_(c)=contact depth (the heightof the contact between the tip and the sample); h_(f)=final depth;S=unloading stiffness.

FIG. 6: Nanoindentation rigs designed to hold the human corneal button(A) and the rat globe (B). The central section of the cornea is locatedby using the microscope, (C), and once located the indenter probe isused, (D).

FIG. 7: Corneal keratocytes seeded in chondrogenic differentiationmedium. Keratocytes cultured for 3 weeks in chondrogenic differentiationmedium containing TGFβ3 and dexamethasone formed spheres, which werelabelled with: (A) nestin around the periphery of the spheres; (B)collagen type II within the core. The culture medium was then switchedto serum containing fibroblast proliferation medium for 1 week, causingcells from the spheres to spread out and populate the dish, (C). Cellsin monolayer were negative for type II collagen whereas the cellclusters remained positive for collagen type II, (D).

FIG. 8: Corneal keratocytes seeded in serum containing fibroblastproliferating medium. Keratocytes cultured in control fibroblastproliferation medium for 3 weeks were negative for nestin (A), andcollagen Type II (B). Confluent fibroblasts were then cultured inchondrogenic differentiation medium containing TGFβ3 and dexamethasonefor 3 weeks, (C). The cells remained negative for collagen type II, (D).(E): pellet culture of confluent fibroblasts in chondrogenicdifferentiation medium. After 3 weeks in culture the cell pellet wassectioned and labelled positive for the keratocyte marker keratocan,(F), and negative for the chondrocyte specific type II collagen, (G).

FIG. 9: Human corneal slices cultured for 2 weeks in control medium (A)and (D) were negative for type II collagen and positive for type Icollagen, respectively. Human corneal slices cultured for 1 week, (B)and (E), and 2 weeks, (C) and (F), in chondrogenic differentiationmedium and labelled for collagen type II, (B) and (C), and type I, (E)and (F). Strong labelling for type II collagen was seen in cornealslices treated for 2 weeks whereas slices treated for only 1 week werenegative for type II collagen. Slices cultured in chondrogenicdifferentiation medium for both the time periods, although less stronglylabelled when compared to the control treated slices, were positive forthe native corneal collagen type I.

FIG. 10: Human corneal slices cultured for 2 weeks in: (A) controlmedium and (B) chondrogenic differentiation medium and labelled forcollagen type II. Similar results were obtained as shown by FIG. 9. Invivo experiments showing (C): untreated corneas; (D) and (E): treatedcorneas with a widespread labelling of type II collagen in the TGFβ3 anddexamethasone treated corneas of rats. Stronger labelling was seen inthe anterior (upper) part of the cornea, (D). Type II collagen appearedfibrillar and was evenly distributed throughout the ECM.

FIG. 11: Keratoconic corneal button cultured in vitro in control medium,(A), (C) and (E), and chondrogenic differentiation medium, (B), (D) and(F), for 2 weeks and labelled for collagen type II (A) and (B), andvimentin (C)-(F), respectively. Compared to the labelling in normalhuman corneas the labelling of type II collagen in treated keratoconiccorneas (B) was weaker. However, the deposition of type II collagen hada similar pattern to that previously seen after in vitro and in vivotreatment of normal human and rat corneas. The fibroblast population inthe treated half of the keratoconic button (D) and (F) increased innumber and the keratocytes appeared healthier and intact with multiple,long cell processes (F), when compared to the untreated half of thekeratoconic cornea, (C) and (E).

FIG. 12: Ex vivo cultured human keratoconic cornea cultured for 3 weeksin control medium, (A) and (C), and chondrogenic differentiation media,(B) and (D), and labelled for alpha smooth muscle actin (αSMA), (A) and(B), and type III collagen, (C) and (D). There was stronger labellingfor αSMA in stromal layer of corneas cultured in control medium (A),when compared to corneas cultured in chondrogenic differentiationmedium. Corneas cultured in either of the two media did not labelpositively for type III collagen.

FIG. 13: Corneal transparency of in vivo treated corneas. Treated, (A)and (C), and untreated, (B) and (D), corneas. After 3 weeks the treatedand untreated corneas were indistinguishable from each other. The frontview of the corneas (A) and (B) reveal a clear cornea through whichlight easily passes to reveal the blood vessels of the back of the eyevery clearly. At 8 weeks the in vivo imaging of the cross section of thecornea reveals a clear, transparent cornea through which light easilypasses. There were no signs of corneal opacity or scarring.

FIG. 14: Quantitative gene expression of collagen type II (A), andcollagen type I (B), in in vivo treated corneas. There was an initialincrease in type II collagen expression upon after 1 week of treatment.Upon withdrawal of the treatment there was a marked decrease in type IIcollagen expression, (A). Native corneal collagen type I expression wasalso initially upregulated, however upon long term treatment (up to 7weeks) its expression was comparable to the control untreated cornea,(B).

FIG. 15: Comparison of 1 week in vivo treated and untreated corneas doesnot reveal a significant difference in hardness (H) and reduced elasticmodulus (Er).

FIG. 16: Load deformation curves obtained for 3 week in vivo treated anduntreated corneas from two rats. The corresponding graphs with theplotted values clearly show an increase in elastic modulus (Er) andhardness (H) in the treated corneas.

FIG. 17: Comparison of elastic modulus and hardness of 8 week treatedand control human keratoconic cornea reveals a marked increase in bothparameters in the treated cornea.

FIG. 18: Reshaping of the cornea in the sheep eye by combining in vivocell reprogramming with a rigid contact lens to hold the desired cornealshape during treatment.

FIG. 19: (A) The Phoenix Micron IV in vivo eye imaging system. Theimaging system enables measurement of corneal thickness, curvature, andtransparency. (B) An OCT attachment enables visualisation of theanterior eye and measurement of corneal thickness and integrity, similarto the image seen here. (C) A nanoindenter and (D) schematicrepresentation of the set-up which will be used to assess cornealbiomechanics ex vivo in sheep. Output is shown as a load-displacementcurve which can be analysed to obtain Young's modulus of elasticity, anda measure of hardness. In large animals such as sheep, corneal thickness(E) is indicated in microns and corneal curvature measurements (F) areobtained using a portable Pentacam®. For corneal curvature (F), widelyspaced colour contours indicate a large radius of curvature; narrowercontours indicate areas of steeper curvature. Numbers indicate theradius of curvature at each point.

FIG. 20: Only TGFβ3 combined with dexamethasone produces collagen typeII deposition. Sheep corneal tissue were cultured in (A) BMP6, (B)BMP6+hydrocortisone, (C) TGFβ3+hydrocortisone, (D) BMP6+dexamethasone,(E) TGFβ3+prednisone, (F) TGFβ3+Triesense®, (G) and (H)TGFβ3+dexamethasone at 20× and 60× magnification, respectively, andlabelled for cartilage specific collagen type II.

FIG. 21: Dose response study for combinations of TGFβ3 anddexamethasone. Sheep corneas were cultured for 3 weeks and labelled forcollagen type II.

DETAILED DESCRIPTION OF THE INVENTION

The following description sets forth numerous exemplary configurations,parameters, and the like. It should be recognised, however, that suchdescription is not intended as a limitation on the scope of the presentinvention, but is instead provided as a description of exemplaryembodiments.

Definitions

In each instance herein, in descriptions, aspects, embodiments, andexamples of the present invention, the terms “comprising”, “including”,etc., are to be read expansively, without limitation. Thus, unless thecontext clearly requires otherwise, throughout the description and theclaims, the words “comprise”, “comprising”, and the like are to beconstrued in an inclusive sense as to opposed to an exclusive sense,that is to say in the sense of “including but not limited to”.

As used herein, “augmenting” refers to methods of increasing one or moreof the thickness, hardness, elastic modulus, tensile strength, andregularity of the cornea, including the corneal tissue (e.g., thestromal layer). Augmentation may be used to impose a particular shape tothe cornea, i.e., corneal curvature. Augmentation methods may beperformed in the presence or absence of a particular condition of theeye, or of the cornea. Augmentation may involve the increase incomponents in the extracellular matrix of the cornea (e.g., collagentype II). Augmentation may also involve increasing the number of cells(e.g., keratocytes) in the cornea. The number of cells may be increased,for example, by altering the proliferative state of such cells fromquiescent to active.

“Co-administration” or “co-administering” refers to the combined use ofagents, for example, therapeutic agents for the eye, and includes theadministration of co-formulations (i.e., combination formulations), aswell as the simultaneous or sequential administration of separateformulations. Similarly, “in conjunction” refers to the combined use ofa therapeutic composition and a therapeutic device/procedure. This caninclude use of the composition preceding use of the device/procedure,simultaneously with the device/procedure, and/or following use of thedevice/procedure.

A “condition” of the cornea refers to a state of disease, defect,damage, injury, degeneration, or dysfunction of the cornea. Thecondition may affect the corneal tissue (e.g., the stromal layer) orcorneal cells (e.g., keratocytes). The condition may be an acutecondition, for example, an abrasion or ulceration, or may be a chroniccondition, for example, keratoconus or myopia.

The “cornea” as used herein refers to the transparent front part of theeye that covers the iris, pupil, and anterior chamber of the eye. Itincludes the corneal epithelium, Bowman's layer, corneal stroma,Descemet's membrane, and the corneal epithelium. Of particular interestis the stromal layer (also called the substantia propria) of the cornea,which comprises an extracellular matrix of regularly arranged collagenfibres along with keratocytes,

A “derivative”, as relating to a chemical derivative, refers to acompound that has been chemically modified. The present disclosureencompasses each of the chemical compounds described herein as well asany derivatives thereof, including chemically modified forms such assalts, hydrides, esters, and other modifications of the originalcompound.

“Isolated” as used herein, with particular reference to polypeptides,refers to a molecule that is separated from its natural environment. Anisolated molecule may be obtained by any method or combination ofmethods as known and used in the art, including biochemical,recombinant, and synthetic techniques. To obtain isolated components,the polypeptides may be prepared by at least one purification orenrichment step. Of particular interest are polypeptides and peptidesobtained by artificial means, i.e., non-natural, means. This includesbut is not limited to, synthetic chemistry, recombinant technology,purification protocols, etc. Included are polypeptides isolated fromnatural, recombinant, or synthetic sources. Also included arepolypeptides produced by chemical synthesis, or by plasmids, vectors, orother expression constructs that may be introduced into a cell orcell-free translation system. Such polypeptides are clearlydistinguished from polypeptides as they naturally occur, without humanintervention.

The terms “protein” or “polypeptide” (e.g., SEQ ID NO:1), and other suchterms, for simplicity, refer to the molecules described herein. Suchterms are not meant to provide the complete characterization of thesemolecules. Thus, a protein or polypeptide may be characterised herein ashaving a particular amino acid sequence, a particular 2-dimensionalrepresentation of the structure, but it is understood that the actualmolecule claimed has other features, including 3-dimensional structure,mobility about certain bonds and other properties of the molecule as awhole. It is the molecules themselves and their properties as a wholethat are encompassed by this disclosure. The terms “protein” and“polypeptide” are used interchangeably herein.

A TGFβ3 “polypeptide” refers to polypeptides obtained from any source,e.g., isolated naturally occurring polypeptides, recombinantpolypeptides, and synthetic polypeptides, and to include polypeptideshaving the naturally occurring amino acid sequence as well aspolypeptides having variant amino acid sequences, and fragments of suchsequences, as described in detail herein. TGFβ3 may also be referred toin the art as transforming growth factor-beta3, TGFB3, ARVD, andFLJ16571.

Amino acid “sequence identity” refers to the amino acid to amino acidcomparison of two or more polypeptides. A test sequence may be identicalto a reference sequence (i.e., share 100% identity), or may include oneor more amino acid substitutions. In preferred aspects, amino acidsubstitutions may possess similar chemical and/or physical propertiessuch as charge or hydrophobicity, as compared to the reference aminoacid. Sequence identity may be typically determined by sequencealignments at the regions of highest homology. Sequence alignmentalgorithms, for example BLAST® sequence alignment programs, are wellknown and widely used in the art. Based on the sequence alignment, thepercent identity can be determined between the compared polypeptidesequences.

A “refractive error” as used herein, refers to error in the focusing oflight by the eye. Refractive errors may include spherical errors andcylindrical errors. Both lower order aberrations and higher orderaberrations are included. Specifically included as refractive errors arethe conditions of the eye noted as myopia, hyperopia, astigmatism,anisometropia, and presbyopia.

“Regeneration”, in relation to the cornea, refers to the restoration ofone or more of the shape, thickness, regularity, hardness, elasticmodulus, and tensile strength of the cornea, including that of thecorneal tissue (e.g., the stromal layer). Methods of regeneration may beused to impose a particular shape to the cornea, i.e., cornealcurvature. Regeneration methods may be performed in the treatment of aparticular condition of the eye, or of the cornea. Regeneration mayinvolve the increase in components in the extracellular matrix of thecornea (e.g., collagen type II). Regeneration may also involveincreasing the number of cells (e.g., keratocytes) in the cornea. Thenumber of cells may be increased, for example, by altering theproliferative state of such cells from quiescent to active.

“Reprogramming” of cells, for example, for corneal cells (e.g.,keratocytes) refers to changes in the state of differentiation.Reprogramming is associated with one or more changes in cell morphology,cellular gene expression (e.g., collagen expression, including collagentype I and/or type II expression), or the cells proliferative state(e.g., quiescent or active).

The term “subject” refers to a human or non-human animal.

“Preventing” refers to stopping or delaying the onset of a condition,for example an eye condition, or particularly a corneal condition, suchas a disorder or other defect of the cornea. A preventative measure willresult in the stoppage or delay of one or more symptoms of thecondition, or a lessening of symptoms if such do arise. Prevention of acorneal condition may involve augmenting the cornea, as described indetail herein.

“Treating” refers to reducing, ameliorating, or resolving a condition,for example an eye condition, or particularly a corneal condition, suchas a disorder or other defect of the cornea. A treatment will result inthe reduction, amelioration, or elimination of one or more symptoms ofthe condition. Treatment of a corneal condition may involve regenerationof the cornea, as detailed herein. The compositions and methods of theinvention may be used for treating various conditions, for preventingvarious conditions, or for both treating and preventing variousconditions, as described in detail herein.

Cell and Tissue Regeneration

Cell and tissue regeneration technologies hold considerable promise intherapeutic treatments. As disclosed herein, the inventors havedeveloped compositions and methods for modulating cells using in situcell reprogramming in order to affect collagen type II expression andextracellular matrix (ECM) deposition in corneal tissue. This, in turn,is used to strengthen and/or augment the cornea of the eye. Theinventors thereby provide a unique approach for the in situ/in vivoregeneration and augmentation of the corneal stromal matrix.

Accordingly, the disclosed methods may be utilised in in vivo tissueengineering therapy for various conditions of the cornea, includingmyopia and keratoconus. As noted above, myopia is characterised by theexcessive curvature of the cornea (FIG. 1A), while keratoconus is aprogressive ectatic corneal dystrophy leading to a characteristicpattern of corneal thinning (FIG. 1B; image adapted from Romero-Jiménez,Santodomingo-Rubido, & Wolffsohn 2010).

Corneal keratocytes are relatively quiescent and normally only producelarge amounts of extracellular matrix (ECM) when they switch to afibroblast or myofibroblast phenotype. ECM deposition associated withthose phenotypes usually leads to corneal fibrosis and loss oftransparency (Kadler, Baldock, Bella, & Boot-Handford 2007).Chondrocytes, the cells that make up cartilage, secrete type II collagenwhich is a fibrillar collagen similar to type I found in the cornea.Type II collagen is also expressed by keratocytes during development ofthe chick cornea and it is only later replaced by type I in the maturechick stroma (Linsenmayer et al. 1990).

The inventors have previously shown that stromal cells from adult humanand rat corneas can be reprogrammed to produce neuron specific proteinswhen treated with neuronal lineage specifying growth factors (Greene etal. 2013). This data demonstrates that an adult cell population can bereprogrammed simply by the modulation of the growth factor environmentboth in vitro and in vivo.

Now, as demonstrated herein, the inventors show that corneal stromalcells can be induced in vitro and ex vivo to produce cartilage specificfibrillar collagen, collagen type II, by treating the cells withtransforming growth factor β3 (TGFβ3) and dexamethasone (Examples 8 and9). In particular, the inventors have demonstrated that keratocytes inhuman keratoconic corneal biopsies express collagen type II when treatedwith these two compounds (Example 8). In addition, with animal studies,the inventors have demonstrated that the two compounds of TGFβ3 anddexamethasone can be delivered in vivo using eye drops to stimulatecollagen II deposition (Example 9). Notably, the deposition of collagentype II was uniform, improving the biomechanics of the cornea, with nofibrosis or scarring, and no effect on corneal transparency (Examples 11and 13).

Without wishing to be bound by theory, it is hypothesised that thecollagen deposition is brought about by the reprogramming of cellswithin the stroma to a chondrocyte phenotype. It is known thatchondrocytes secrete type II collagen which is not only a fibrillarcollagen similar to type I found in the cornea, but is also expressedduring development of the chick cornea (Linsenmayer et al. 1990). It isonly later replaced by collagen type I in the mature stroma (Linsenmayeret al. 1990).

In the results described herein, an initial increase in collagen type Iexpression was observed upon treatment of corneal keratocytes with TGFβ3and dexamethasone (Example 12). However, the inventors consider that theobserved level of collagen type I deposition would be insufficient tostiffen/reshape a cornea. Furthermore, the deposition of collagen typeII is deemed more feasible as a treatment strategy. It is noted thatcollagen type II is less susceptible to enzymatic degradation, forexample, by enzymes present in a keratoconic cornea.

In accordance with the inventors' results, it is possible to use thereprogramming of keratocytes to produce new ECM molecules as aneffective treatment to improve the biomechanical characteristics of thecornea. This approach is considered advantageous, as it reducessusceptibility to degradation by corneal enzymes, as noted above. Thedisclosed treatment module aims not only to stabilise the cornea, butalso to provide remedial aid for conditions of the eye, includingvarious corneal conditions and refractive errors of the eye. Thus, themethods of the invention may be used, for example, for the treatment ofkeratoconic keratocytes in the ectatic cornea. Additionally, the methodsof the invention may be used for the treatment of myopia and variousother conditions of the cornea, as described in detail herein.

Conditions Affecting the Eye and Cornea

The compositions described herein find particular use in regenerating oraugmenting the cornea (e.g., the stromal layer), as well as cornealcells (e.g., keratocytes). The compositions may be used to addresscorneal thinning, weakening, cell loss, tissue loss, matrix loss,collagen loss, and/or irregularity. In this way, the compositionsdescribed herein may be utilised for various conditions affecting theeye, including conditions involving corneal defects, disease, damage,injury, and/or degeneration, as well as refractive errors of the eye.

In specific aspects, the invention encompasses methods for treatingdefects of the cornea. In certain situations, the methods of theinvention may also be used to prevent corneal defects. The defects maybe associated with a particular condition of the cornea. Exemplaryconditions include keratoconus, as described in detail herein, andrelated conditions, which include corneal ectasias such as keratoglobus,pellucid marginal degeneration, and posterior keratoconus (see, e.g.,Arffa 1997; Krachmer et al. 1984; Rabonitz 2004; Jinabhai et al. 2010).Specifically included as defects are myopia, presbyopia, and alsoastigmatisms, which encompass regular and irregular astigmatisms.Congenital defects of the cornea are also included. Amongst these arecornea plana and microcornea, the latter of which may be associated withfetal alcohol syndrome, Turner syndrome, Ehlers-Danlos syndrome,Weill-Marchesani syndrome, Waardenburg's syndrome, Nance-Horan syndrome,and Cornelia de Lange's syndrome. Included also is keratoglobus(mentioned above) that may be associated with Ehlers-Danlos syndrometype IV.

In further aspects, the invention encompasses methods for treatingdamage or degeneration of the cornea. In certain situations, the methodsof the invention may be used to prevent corneal damage. Damage ordegeneration may be associated with a particular condition of thecornea. Specifically included is corneal melt, for example, corneal meltassociated with an inflammatory disorder, such as rheumatoid arthritis.Other exemplary conditions include keratitis, such as marginalkeratitis, stromal keratitis, exposure keratitis, neurotrophickeratitis, filamentary keratitis, rosacea keratitis, viral keratitisincluding herpes keratitis, fungal keratitis, protozoal keratitis, andother infectious keratitis, such as luetic interstitial keratitis,microsporidial keratitis, Thygeson's keratitis, and infectiouscrystalline keratopathy. Included also is ulcerative keratitis, alsocalled peripheral ulcerative keratitis (PUK), which includes ulcerativekeratitis that is associated with a systemic disease, such as rheumatoidarthritis Wegener's granulomatosis, systemic lupus erythematosus,relapsing polychondritis, and polyarteritis nodosa. Endophthalmitis isalso included. Included as well are chronic corneal edema, Mooren'sulcer, dellen, phlyctenulosis, Terrien's degeneration, Salzman'sdegeneration, spheroidal degeneration, and Fuch's dystrophy. Suchconditions are well known and well characterised in the art. See, e.g.,Jackson 2008; Denniston 2009; and Willshaw et al. 2000. Additionallyincluded are stromal dystrophies, for example, lattice corneal dystrophy(e.g., type 1 and type 2), granular corneal dystrophy (e.g., type 1 andtype 2), macular corneal dystrophy, Schnyder corneal dystrophy,congenital stromal corneal dystrophy, and fleck corneal dystrophy.

In still further aspects, the invention encompasses methods for treatinginjury to the cornea. Included are injuries due to physical damage,chemical damage, radiation damage, and/or damage from particularmedication. Injury may be associated with corneal abrasion, cornealerosion, corneal puncture, membrane rupture, corneal scarring, orcorneal ulcers, including melting ulcers, indolent ulcers, andsuperficial ulcers. Included also are injuries and other damageassociated with eye surgery, including surgical wounds, corneal damagefollowing radial keratectomy, and acute problems following keratoplasty,which include persistent epithelial defects. Additionally included areinjuries associated with corneal melt, for example, corneal meltfollowing surgery or other treatments of the eye (e.g., topical NSAIDadministration). Corneal melting may be attributable to infectious,inflammatory, or trophic causes. Included also are injuries and damageof the cornea associated with aging.

In even further aspects, the invention encompasses methods for treatingor preventing refractive errors of the eye. Such refractive errors maybe associated with particular conditions, including myopia, hyperopia,presbyopia, anisometropia, higher order aberrations, and variousastigmatisms. Higher order aberrations include, but are not limited to,comas, trefoils, quadrafoils, spherical aberrations, and aberrationsidentified by mathematical expressions (e.g., Zernike polynomials).

Conditions of the cornea may be diagnosed by various methods, includingfluorescein staining, which may include a Seidel's test, specularmicroscopy, corneal topography, isometric tomography, pachymetry,ultrasound, slit lamps, corneal scrapes, and biopsies. Diagnosis mayalso involve assessments for visual acuity and/or opacification. Cornealconditions may be associated with one or more symptoms of: pain,photophobia, foreign body sensation, reduced visual acuity, oedema,white cell infiltrate, fluorescein uptake, vascularisation, redness, andsystemic symptoms such as headaches, nausea, and fatigue. Similarly,symptoms of refractive errors may include but are not limited to:reduced visual acuity as well as blurry vision, double vision, hazinessof vision, visual fatigue, foreign body sensation, problematic glare orhalos, starburst patterns, ghost images, impaired night vision,squinting, excessive staring, excessive blinking, headaches, eyerubbing, eye strain, eye surface dessication, eye irritation, redness,and spasms of the eye.

Therapeutic Compositions

As noted above, the compositions described herein may be utilised fortreating and/or preventing various conditions of the eye, includingconditions affecting the cornea and refractive errors of the eye. Thecompositions may include a TGFβ3 polypeptide, or variants or fragmentsthereof, along with dexamethasone, or derivatives thereof or relatedsteroidal agents.

In various aspects, the composition may be formulated to include thenoted combination of components (a TGFβ3 polypeptide (or variants orfragments thereof) plus dexamethasone (or derivatives thereof or relatedsteroidal agents)), or may be formulated to include a first component (aTGFβ3 polypeptide (or variants or fragments thereof) or alternativelydexamethasone (or derivatives thereof or related steroidal agents)) withthe second component to be added in prior to administration.Alternatively, the composition may be formulated to include a firstcomponent (a TGFβ3 polypeptide (or variants or fragments thereof) oralternatively dexamethasone (or derivatives thereof or related steroidalagents)), which is used in simultaneous or sequential administrationwith a formulation that includes the second component.

In one aspect, the TGFβ3 polypeptide may include at least the followingamino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYYANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQLSNMVVKSCK CS (SEQ ID NO:1) (GenBank Reference CAR70088.1). The TGFβ3polypeptide may include at least 112 amino acids shown above, and mayhave a molecular mass of 25.5 kDa. Alternatively, the TGFβ3 polypeptidemay be derived from amino acids 644-850 (207 amino acids) of theprecursor polypeptide sequence identified in GenBank ReferenceCAA33024.1; GenBank Accession No. CAA33024; or NCBI Reference SequenceNP_003230.1.

In other aspects, a TGFβ3 variant or fragment may be utilised. Forexample, the variant or fragment may exhibit at least 75% sequenceidentity to SEQ ID NO:1, preferably at least 80% identity, morepreferably at least 85%, most preferably at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or about 100% sequence identity toSEQ ID NO:1, as described herein. It is of particular interest where theTGFβ3 variant exhibits biological activity, for example, activity thatis similar or improved compared to the non-variant polypeptide

In further aspects, a particular fragment may be utilised. For example,the TGFβ3 fragment may comprise at least 80 amino acids, at least 85amino acids of SEQ ID NO:1, more preferably at least 90 amino acids, atleast 91 amino acids, at least 92 amino acids, at least 93 amino acids,at least 94 amino acids, at least 95 amino acids, at least 96 aminoacids, at least 97 amino acids, at least 98 amino acids, at least 99amino acids, most preferably at least 100 amino acids, at least 101amino acids, at least 102 amino acids, at least 103 amino acids, atleast 104 amino acids, at least 105 amino acids, at least 106 aminoacids, 107 amino acids, 108 amino acids, 109 amino acids, 110 aminoacids, or 111 amino acids of SEQ ID NO:1. Of particular interest arefunctional fragments of TGFβ3, for example, fragments that exhibitbiological activity, for example, activity that is similar or improvedcompared to the reference polypeptide.

In a particular aspect, the TGFβ3 polypeptide or the variant or fragmentthereof may be provided as a recombinant polypeptide. For example, thepolypeptide may be expressed in cell or cell-free expression systems aswidely known and used in the art. Included amongst these are bacterial,fungal, plant, and mammalian expression systems. Expression systemsusing E. coli cells, CHO cells, HEK cells, and Nicotiana benthamianacells are specifically included. The TGFβ3 polypeptide or the variant orfragment thereof may be provided as a human recombinant polypeptideexpressed in human or non-human expression systems. The TGFβ3polypeptide or the variant or fragment thereof may be provided as adisulfide-linked homodimeric, non-glycosylated, polypeptide chain, inaccordance with known methods.

The TGFβ3 polypeptide or the variant or fragment thereof may be isolatedfrom recombinant expression systems by standard methods, including wellknown chromatographic techniques. The TGFβ3 polypeptide or the variantor fragment thereof may include a sequence tag to facilitate cleavage,isolation, and/or localisation of the polypeptide. In accordance withthe present invention, the TGFβ3 polypeptide may be obtained fromvarious commercial sources. For example, recombinant human TGFβ3 may beobtained from R&D Systems (Catalogue Nos. 243-B3-002; 243-B3-010),BioVision, Inc. (Catalogue Nos. 4344-500; 4344-50; 4344-5), or ProspecProtein Specialists (Catalogue Nos. CYT-113; CYT-319).

The biological activity of the TGFβ3 polypeptide or the variant orfragment thereof may be measured in accordance with widely known andused methods. For example, biological activity may be measured inculture by the polypeptides ability to inhibit the mink lung epithelial(Mv1Lu) cells proliferation (see, e.g., Premaraj et al. 2006). Exemplaryactivity by this measurement is shown by an ED₅₀ of ≦50 ng/ml.Alternatively, biological activity may be measured by the dose-dependentinhibition of IL-4 induced proliferation of mouse HT-2 cells (BALB/cspleen activated by sheep erythrocytes in the presence of IL-2) (see,e.g., Tsang et al. 1995). Exemplary activity by this measurement istypically 0.1 to 0.5 ng/ml. Alternatively, the composition that includesthe combination of agents described herein may be measured forbiological activity using the methods noted below. For example,induction of collagen type II (e.g., collagen type II, alpha 1) inkeratocytes can be assessed by one or more of: immunohistochemicalassays, protein assays, Western blot analysis, polymerase chain reaction(PCR) analysis, and quantitative PCR technologies.

As described herein, the composition may also include dexamethasone, aderivative thereof, and/or a related steroidal agent. Dexamethasone ischaracterised as having the following chemical structure:

The trade names for dexamethasone include, for example, Decadron®,Dexasone®, Diodex®, Hexadrol®, Maxidex®, and Minims®.

In various aspects, derivatives of dexamethasone may be used, includingany esters and salts thereof. Exemplary derivatives include but are notlimited to: dexamethasone-17-acetate (CAS RN: 1177-87-3), dexamethasonedisodium phosphate (CAS RN: 2392-39-4), dexamethasone valerate (CAS RN:14899-36-6), dexamethasone-21-isonicotinate (CAS RN: 2265-64-7),dexamethasone palmitate (CAS RN: 33755-46-3), dexamethasone propionate(CAS RN: 55541-30-5), dexamethasone acefurate (CAS RN: 83880-70-0),dexamethasone-21-galactoside (CAS RN: 92901-23-0), dexamethasone21-thiopivalate, dexamethasone 21-thiopentanoate, dexamethasone21-thiol-2-methyl-butanoate, dexamethasone 21-thiol-3-methyl-butanoate,dexamethasone 21-thiohexanoate, dexamethasone21-thiol-4-methyl-pentanoate, dexamethasone21-thiol-3,3-dimethyl-butanoate, dexamethasone21-thiol-2-ethyl-butanoate, dexamethasone 21-thiooctanoate,dexamethasone 21-thiol-2-ethyl-hexanoate, dexamethasone21-thiononanoate, dexamethasone 21-thiodecanoate, dexamethasone21-p-fluorothiobenzoate or a combination thereof. Specifically includedare dexamethasone alcohol and dexamethasone sodium phosphate.Dexamethasone derivatives are also included, as described in U.S. Pat.No. 4,177,268.

The composition may include related steroidal agents, in lieu of or inaddition to dexamethasone. For example, other corticoid steroids may beutilised, in replacement of or along with dexamethasone. Preferred foruse as related steroids are Group C steroids according to Coopmanclassification, which includes betamethasone-type steroids, such asdexamethasone, dexamethasone sodium phosphate, betamethasone,betamethasone sodium phosphate, and fluocortolone. Other relatedsteroidal agents include but are not limited to: fluoromethalone,lotoprendol, medrysone, prednisolone, prednisone, rimexolone,hydrocortisone, lodoxamide, or any derivative or combination thereof.Specifically included are fluoromethalone acetate, fluoromethalonealcohol, prednisolone acetate, prednisolone sodium phosphate,lotoprendol etabonate, hydrocortisone acetate, and lodoxamidetromethamine. It is understood, for any of the chemicals of thisdisclosure, that the chemicals may be in various modified forms such asacetate forms, and sodium phosphate forms, sodium salts, and the like.

The composition may include, for example, 0.04 ng/ml to 4 ng/ml; or 0.04ng/ml to 0.4 ng/ml; or 0.4 ng/ml to 4 ng/ml; or 4 to 40 ng/ml; or 40ng/ml to 400 ng/ml, or 40 ng/ml to 4000 ng/ml dexamethasone, orderivative thereof or related steroidal agent; or about 0.04 ng/ml,about 0.08 ng/ml, about 0.12 ng/ml, about 0.4 ng/ml, about 0.8 ng/ml,about 1.2 ng/ml, about 4 ng/ml, about 12 ng/ml, about 24 ng/ml, about 40ng/ml, about 80 ng/ml, about 120 ng/ml, about 240 ng/ml, about 400ng/ml, about 800 ng/ml, about 1000 ng/ml, about 1600 ng/ml, about 2000ng/ml, about 2400 ng/ml, about 3200 ng/ml, or about 4000 ng/mldexamethasone, or derivative thereof or related steroidal agent.

As further examples, the composition may include 0.4 μg/ml to 40 μg/ml;or 0.4 μs/ml to 4 μg/ml; or 4 μg/ml to 40 μg/ml dexamethasone, orderivative thereof or related steroidal agent; or about 0.4 μg/ml, about0.8 μg/ml, about 1 μg/ml, about 1.2 μs/ml, about 2 μg/ml, about 4 μg/ml,about 8 μg/ml, about 12 μg/ml, about 20 μg/ml, or about 40 μg/mldexamethasone, or derivative thereof or related steroidal agent.

As yet further examples, the composition may include 0.1 mg/ml to 1mg/ml; or 0.5 mg/ml to 5 mg/ml; or 1 mg/ml to 10 mg/ml; dexamethasone,or derivative thereof or related steroidal agent; or about 0.1 mg/ml,about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml, or about10 mg/ml dexamethasone, or derivative thereof or related steroidalagent.

The composition may include, for example, 1 ng/ml to 1 μg/ml; or 1 ng/mlto 10 ng/ml; or 10 ng/ml to 100 ng/ml; or 100 ng/ml to 1 μg/ml TGFβ3polypeptide or variants or fragments thereof, or about 1 ng/ml, about 5ng/ml, about 10 ng/ml, about 20 ng/ml, about 50 ng/ml, about 100 ng/ml,about 200 ng/ml, about 500 ng/ml, about 800 ng/ml, or about 1 μg/mlTGFβ3 polypeptide or variants or fragments thereof. In particularaspects, the composition may include at least 40 ng/ml dexamethasone, orderivative thereof or related steroidal agent, along with at least 4ng/ml TGFβ3 polypeptide, or variants or fragments thereof.

The composition may also include one or more anti-inflammatory agents.Exemplary anti-inflammatory agents include, at least, ketotifenfumarate, diclofenac sodium, flurbiprofen sodium, ketorlac tromethamine,suprofen, celecoxib, naproxen, rofecoxib, or any derivative orcombination thereof. Particularly included are non-steroidalanti-inflammatory drugs (NSAIDs). The composition may additionallyinclude one or more anaesthetic agents. Exemplary anaesthetics include,at least, topical anaesthetics such as proparacaine, lidocaine, andtetracaine, and any derivative or combination thereof. Other agents forthe eye may be selected for inclusion with the composition; these may bechosen by the skilled artisan based on the condition and needs of thesubject under treatment.

The compositions as described herein may be formulated for topicaladministration, as described herein and in accordance with knownmethods. In certain circumstances, intraocular administration may bedesirable. The composition may be provided in any form suitable foradministration to the eye. Exemplary formulations include, at least,solutions, suspensions, emulsions (dispersions), gels, creams, orointments in a suitable ophthalmic vehicle. For example, the compositionmay be provided in the form of eye drops, a semisolid gel, or a spray.In certain aspects, moulding contact lenses or other inserts/implantsmay be impregnated with the composition of the invention. In thismanner, the composition can be delivered to the cornea continuously andin a time-release manner as the subject is wearing the contact lenses.

For topical administration to the eye, the compositions may beformulated with a pH range of 5.0 to 8.0. This pH range may be achievedby the addition of buffers to the solution. It is preferred that theformulations are stable in buffered solutions. That is, there is noadverse interaction between the buffer and the active agents that wouldcause the composition to be unstable, e.g., by precipitation oraggregation. The composition may be hypertonic (5% to 40%, preferablyapproximately 10, 20, 30, or 40%) or hypotonic (0% to 5%, preferablyapproximately 1, 2, 3, or 4%) depending on the needs of the subject(e.g., working needs, rest hours, sleeping, etc.) A hypertoniccomposition (e.g., 40%) may be used when combined with moulding contactlenses, as described in detail herein.

The compositions may include one or more suitable preservatives asoptional ingredients. Suitable preservatives may be added to preventcontamination, for example, bacterial contamination. Such agents mayinclude, but are not limited to, benzalkonium chloride, thimerosal,chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol,EDTA, sorbic acid, Onamer® M, and other agents known to those skilled inthe art, or any combination thereof. Such preservatives may be typicallyemployed at a level of 0.001% to 1.0% by weight of the composition.

The compositions may contain an optional co-solvent. The solubility ofthe components of the present compositions may be enhanced by asurfactant or other appropriate co-solvent in the composition. Suchco-solvents/surfactants include, for example, polysorbate 20, 60, and80, polyoxyethylene/polyoxypropylene surfactants (e.g. Pluronic® F-68,F-84, and P-103), cyclodextrin, tyloxapol, and other agents known tothose skilled in the art, and any combination thereof. Such co-solventsmay be typically employed at a level of 0.01% to 2% by weight of thecomposition.

Penetration enhancing agents may be used to increase uptake of thecomposition into the eye. Exemplary agents include, at least,cetylpyridinium chloride, ionophores such as lasalocid, benzalkoniumchloride, Parabens, Tween 20, saponins, Brij 35, Brij 78, Brij 98,ethylenediaminetetraacetic acid, bile salts, and bile acids (such assodium cholate, sodium taurocholate, sodium glycodeoxycholate, sodiumtaurodeoxycholate, taurocholic acid, chenodeoxycholic acid, andursodeoxycholic acid), capric acid, azone, fusidic acid, hexamethylenelauramide, saponins, hexamethylene octanamide, and decylmethylsulfoxide.

In addition, bioadhesive polymers may be used to adhere to the mucincoat covering the eye, to prolong contact of the composition with theeye. Bioadhesive polymers may be macromolecular hydrocolloids withnumerous hydrophilic functional groups, such as carboxyl-, hydroxyl-,amide, and sulphate capable of establishing electrostatic interactions.Exemplary agents include, at least, polyarylic acid (e.g., carbopol,carbophil, and polycarbophil) and carboxymethyl cellulose.

Controlled release systems may also be used; such systems may involve insitu gels, colloidal particles, nanoparticles, and/or niosomes. Otherdrug delivery systems include but are not limited to: non-erodibleocular inserts, erodible ocular inserts, hydrogels, collagen shields,liposomes, drug-loaded films (e.g., NOD®), and ionotophoresis.

The compositions may include, also, an optional agent to increaseviscosity. Viscosity increased above that of simple aqueous solutionsmay be desirable to increase ocular absorption of the active compounds,to decrease variability in dispensing the formulation, to decreasephysical separation of components of a suspension or emulsion of theformulation and/or to otherwise improve the ophthalmic formulation. Suchviscosity builder agents include as examples polyvinyl alcohol,polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, other agents known to those skilled in the art, or acombination thereof. Such agents may be typically employed at a level of0.01% to 2% by weight of the composition.

In particular aspects, the compositions include a gelling agent, forexample, high molecular weight water-soluble polysaccharides such asgellan gum. Gellan gum may be obtained from various commercial sources,for example, as sold under the trade name Kelcogel®. In particular,Kelcogel® LT100 may be used as a fine mesh, high acyl gellan, whichforms soft, elastic, non-brittle gels. In specific aspects, the gellangum based composition is formulated as a 0.5% eye drop

Other agents may be used to further stabilise or otherwise enhance thecomposition. For example, one or more of EDTA, sodium chloride,tyloxapol, sodium sulfate, and/or hydroxyethylcellulose may haveadditional beneficial effects of further stabilising the composition.

Therapeutic Methods

As noted above, the compositions described herein find particular use inregenerating or augmenting the cornea (e.g., the stromal layer), as wellas corneal cells (e.g., keratocytes). In particular, the compositionsmay be used to provide enhanced shaping, thickness, regularity,hardness, elastic modulus, tensile strength, or functionality (e.g.,refraction) of the cornea. Thus, the compositions described herein maybe used to address various conditions of the cornea and correctrefractive errors of the eye, and may be used as adjunct therapy withother eye treatments.

As previously noted, the composition may be formulated in any suitablemeans for administration to the eye. Included as formulations areophthalmic solutions, creams, emulsions, ointments, and gels.Specifically noted are formulations that are made as eye drops. In aparticular aspect, the composition may be administered as an eye dropusing any of the many types of eye drop dispensers on the market. Asexemplifications, the container for the compositions of the inventionmay be clear, translucent, and opaque and may contain other propertiesor combination of properties such as being glass lined, tamper proof,packaged in single or few dose aliquots, and any combination thereof.

The composition may be administered in therapeutically effective amountsto a subject to achieve a desired medical outcome. In particular, thecomposition may be administered in amounts to address an ophthalmiccondition described herein, or at least mitigate one or more symptoms ofsuch condition. The precise dosage of the composition (i.e., amount andscheduling) may be determined by a clinician, based on the subject andthe condition presented. Exemplary formulations (e.g., eye drops) may beadministered 1 to 24 times per day, or 1 to 12 times per day, or 1 to 6times per day, or 1 to 4 times per day, or 1 to 3 times per day, or 1 to2 times per day, or 1, 2, 3 4, 6, 8, 12, 18, or 24 times per day. Thecomposition may be topically applied as an eye drop by placing one dropin each eye to be treated. Alternatively, 2 to 3 drops may be applied toeach eye.

For the described composition, the dosage range may be, for example, 0.2pg to 2.4 ng; or 2 pg to 2.4 ng of dexamethasone, or derivative thereofor related steroidal agent; or about 0.2 pg, about 0.4 pg, about 0.6 pg,about 0.8 pg, about 1.2 pg, about 2.4 pg, about 2 pg, about 4 pg, about6 pg, about 8 pg, about 12 pg, about 18 pg, about 24 pg, about 0.2 ng,about 0.26 ng, about 0.4 ng, about 0.6 ng, about 0.8 ng, about 1.2 ng,about 1.8 ng, or about 2.4 ng of dexamethasone, or derivative thereof orrelated steroidal agent, per eye for one dose.

As other examples, the dosage range may be 12 ng to 1.3 μg; or 6 ng to600 ng dexamethasone, or derivative thereof or related steroidal agent;or about 6 ng, about 8 ng, about 12 ng, about 16 ng, about 18 ng, about24 ng, about 26 ng, about 30 ng, about 36 ng, about 40 ng, about 48 ng,about 52 ng, about 54 ng, about 60 ng, about 72 ng, about 78 ng, about80 ng, about 90 ng, about 120 ng, about 130 ng, about 160 ng, about 180ng, about 240 ng, about 260 ng, about 300 ng, about 360 ng, about 400ng, about 480 ng, about 520 ng, about 540 ng, about 600 ng, about 720ng, about 780 ng, about 900 ng, about 1.2 μg, about 1.3 μg ofdexamethasone, or derivative thereof or related steroidal agent, per eyefor one dose.

As still other examples, the dosage range may be 1.5 μg to 150 μg; 2.6μg to 260 μg; or 6.5 μg to 650 μg of dexamethasone, or derivativethereof or related steroidal agent; or about 1.5 μg, about 2 μg, about 3μg, about 4.5 μg, about 6 μg, about 6.5 μg, about 7.5 μg, about 10 μg,about 15 μg, about 22.5 μs, about 32.5 μg, about 20 μg, about 26 μg,about 30 μg, about 40 μg, about 45 μg, about 60 μg, about 65 μg, about75 μg, about 80 μg, about 90 μg, about 100 μg, about 120 μg, about 130μg, about 150 μg, about 180 μg, about 225 μg, about 240 μg, about 260μg, about 200 μg, about 300 μg, about 325 μg, about 450 μg, about 600μg, or about 650 μg dexamethasone, or derivative thereof or relatedsteroidal agent, per eye for one dose. It will be recognised thatspecific formulations of dexamethasone are commercially available, andsuch may be utilised in accordance with accepted dosage amounts andscheduling.

Any of the above noted dosages of dexamethasone may be co-administeredwith a dosage range of, for example, 5 pg to 65 ng; or 0.5 ng to 65 ngof TGFβ3 polypeptide, or variants or fragments thereof; or about 5 pg,about 10 pg, about 15 pg, about 20 pg, about 30 pg, about 45 pg, about60 pg, about 0.05 ng, about 0.1 ng, about 0.15 ng, about 0.2 ng, about0.3 ng, about 0.45 ng, about 0.5 ng, about 0.6 ng, about 0.65 ng, about1 ng, about 1.5 ng, about 2 ng, about 3 ng, about 4 ng, about 4.5 ng,about 6 ng, about 6.5 ng, about 7.5 ng, about 9 ng, about 10 ng, about12 ng, about 13 ng, about 15 ng, about 16 ng, about 20 ng, about 22.5ng, about 24 ng, about 30 ng, about 32.5 ng, about 36 ng, about 40 ng,about 45 ng, about 48 ng, about 50 ng, about 52 ng, about 60 ng, orabout 65 ng of TGFβ3 polypeptide, or variants or fragments thereof, pereye for one dose.

Dosage for one eye may be about one drop of the disclosed composition.One drop of composition may be 10 μl to 200 μl, 20 μl and 120 μl, or 50μl to 80 μl or any values in between. For example, dispensers such aspipettors can dispense drops from 1 μl to 300 μl and any value inbetween. Preferably, the dispenser metes out about 15 μl, about 20 μl,about 30 μl, about 45 μl, about 60 μl, or about 65 μl per drop of thedisclosed composition.

Where the composition is administered via a contact lens or anotherinsert/implant device, the contact lens or insert/implant may include,for example, 0.01 mg to 10 mg of dexamethasone, or derivative thereof orrelated steroidal agent; or about 0.01 mg, about 0.1 mg, about 0.5 mg,about 0.7 mg, about 1 mg, about 5 mg, or about 10 mg of dexamethasone,or derivative thereof or related steroidal agent. Alternatively, thecontact lens or insert/implant may include 10 ng to 100 ng ofdexamethasone, or derivative thereof or related steroidal agent; orabout 1 ng, about 5 ng, about 10 ng, about 20 ng, about 50 ng, about 80ng, or about 100 ng dexamethasone, or derivative thereof or relatedsteroidal agent. As further examples, the contact lens or insert/implantmay include about 10 ng to 1 μg of TGFβ3 polypeptide, or variants orfragments thereof; or about 10 ng, about 50 ng, about 100 ng, about 200ng, about 500 ng, about 800 ng, or about 1 μg TGFβ3 polypeptide, orvariants or fragments thereof.

The compositions described herein may be used in conjunction withvarious surgical procedures or other treatments. For example, thecompositions can be used along with surgical and non-surgical methodsfor the refractive correction of the eye. Exemplary methods include butare not limited to: radial keratotomy (RK), including mini asymmetricradial keratotomy (MARK), hexagonal keratotomy (HK), photorefractivekeratectomy (PRK), keratomilleusis, laser in situ keratomileusis(LASIK), e.g., intraLASIK®, laser epithelial keratomileusis (LASEK),e.g., Epi-LASEK, automated lamellar keratoplasty (ALK), laser thermalkeratoplasty (LTK), conductive keratoplasty (CK), limbal relaxingincisions (LRI), astigmatic keratotomy (AK), epikeratophakia, anteriorciliary sclerotomy (ACS), scleral reinforcement surgery, presbyopiareversal, laser reversal of presbyopia (LRP), intracorneal rings (ICR),intrastromal corneal ring segments (e.g., INTACTS®), implantable contactlenses, scleral expansion bands (SEB), and Kamra™ inlays. Also includedare thermokeratoplasty, orthokeratology, enzyme orthokeratology, andchemical orthokeratology.

The compositions may be used in conjunction with surgical correction ofnon-refractive conditions, for example, surgical correction of a cornealtear. In particular aspects, the compositions described herein may beused in conjunction with specific surgical methods performed on thecornea. Exemplary methods include but are not limited to: cornealtransplant surgery, penetrating keratoplasty (PK), phototherapeutickeratectomy (PTK), pterygium excision, corneal tattooing,keratoprosthesis insertion (e.g., KPro or Dohlman-Doane), andosteo-odonto-keratoprosthesis insertion (OOKP).

The compositions may be used in conjunction with corneal collagencrosslinking. Corneal crosslinking typically involves the use ofriboflavin solution activated by exposure to UV-A light. Notedcrosslinking methods include but are not limited to: cornealcrosslinking with the epithelium removed (Dresden protocol, or epi-off),transepithelial crosslinking (epi-on), and accelerated crosslinking.Crosslinking procedures are generally available, and marketed as CXL,C3-R® CCL® and KXL® corneal crosslinking, amongst others. Administrationof the composition may be prior to, and/or subsequent to, thecrosslinking procedure. It is proposed that the disclosed compositionscan be used to avoid or counter the deleterious effects of crosslinkingprocedures, such as stromal haze and cell loss (described in moredetail, below). Moreover, corneal regeneration with the disclosedcompositions can allow crosslinking to be performed on subjects who werepreviously ineligible for such procedures, e.g., those with cornealthickness less than 400 μm. Furthermore, the disclosed compositions canbe used to slow or halt progressive corneal thinning, which would not beaddressed by the use of crosslinking on its own.

The compositions described herein may be co-administered with one ormore additional agents for the eye. In various aspects,co-administration may be by simultaneous or subsequent administrationwith such agents, or by co-formulation with such agents. Depending onthe condition being treated or prevented, the compositions describedherein may be co-administered with one or more agents, which include butare not limited to: antihistamines, sympathomimetics, beta receptorblockers, parasympathomimetics, parasympatholytics, prostaglandins,nutrients, vasoconstrictors, lubricants, anti-microbials, andanaesthetics. Specifically included are various anti-inflammatoryagents, including non-steroidal anti-inflammatory drugs (NSAIDs). Thecompositions may also be co-administered with eye lubricating solutionsand tear-replacing solutions.

Non-limiting examples of anaesthetics include: benzocaine, bupivacaine,cocaine, etidocaine, lidocaine, mepivacaine, pramoxine, prilocalne,chloroprocaine, procaine, proparacaine, ropicaine, and tetracaine.Non-limiting examples of anti-inflammatory agents include: aspirin,acetaminophen, indomethacin, sulfasalazine, olsalazine, sodiumsalicylate, choline magnesium trisalicylate, salsalate, diflunisal,salicylsalicylic acid, sulindac, etodolac, tolmetin, diclofenac,ketorolac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen,suprofen, oxaproxin, mefenamic acid, meclofenamic acid, oxicams,piroxicam, tenoxicam, pyrazolidinediones, phenylbutazone,oxyphenthatrazone, pheniramine, antazoline, nabumetone, COX-2 inhibitors(Celebrex®), apazone, nimesulide, and zileuton. Glucocorticoids such ashydrocortisone, prednisolone, fluorometholone, and dexamethasone mayalso be used as anti-inflammatory agents.

Exemplary anti-microbial agents include but are not limited to:bacitracin zinc, chloramphenicol, chlorotetracycline, ciprofloxacin,erythromycin, gentamicin, norfloxacin, sulfacetamide, sulfisoxazole,polymyxin B, tetracycline, tobramycin, idoxuridine, trifluridine,vidarabine, acyclovir, foscarnet, ganciclovir, natamycin, amphotericinB, clotrimazole, econazole, fluconazole, ketoconazole, miconazole,flucytosine, clindamycin, pyrimethamine, folinic acid, sulfadiazine, andtrimethoprim-sulfamethoxazole. Exemplary vasoconstrictors include butare not limited to: dipivefrin (Propine®), epinephrine, phenylephrine,apraclonidine, cocaine, hydroxyamphetamine, naphazoline,tetrahydrozoline, dapiprazole, betaxolol, carteolol, levobunolol,metipranolol, and timolol. Nutrients include vitamins, minerals, andother beneficial agents such as vitamin A, vitamin B₁, vitamin B₆,vitamin B₁₂, vitamin C (ascorbic acid), vitamin E, vitamin K, and zinc.

In specific aspects, the composition described herein is formulated aseye drops, and such eye drops are used in conjunction with other eyedrop formulations. Such other eye drops may include but are not limitedto: rinse/lubricating eye drops, dry eye treatments, steroid andantibiotic eye drops, glaucoma eye drops, allergy/anti-inflammatory eyedrops, and conjunctivitis eye drops.

The compositions may be used in conjunction with contact lenses, cornealinserts, corneal implants, or intrastromal rings, to assist insupporting or reshaping the subject's cornea. Included amongst cornealinserts are corneal inlay and corneal onlay devices. For example,contact lenses, intrastromal rings, or other inserts/implants may beused for moulding or holding corneal shape preceding, during, and/orfollowing treatment with the composition. It is noted that a corneal‘insert’ typically refers to a temporary device inserted into thecornea, while a corneal ‘implant’ typically refers to a more permanentdevice. However, many well known devices are described interchangeablyin the art as implants/inserts. Therefore, the terms ‘insert/implant’ asused herein are not to be deemed as strictly limiting based on time ofusage.

The contact lens, corneal insert, corneal implant, or intrastromal ringmay be used with the disclosed composition for treatment of cornealdefects, diseases, damage, injury, and/or degeneration, as well asrefractive errors of the eye. In various aspects, the contact lens,corneal insert, corneal implant, or intrastromal ring may act as acarrier for the composition or as a composition eluting device. In otheraspects, the contact lens, intrastromal ring, or other cornealinsert/implant may be utilised with the composition that is suitable foradministration to the eye, e.g., eye drops, as described in detailherein. In certain aspects, computer software may be used to determinethe contact lenses, corneal inserts, corneal implants, or intrastromalrings that are most suitable for the subject and/or to determine theformulation of the composition. In particular aspects, treatmentutilising contact lenses, corneal inserts, corneal implants, orintrastromal rings along with the composition described herein is usedpreceding or following eye surgery, e.g., refractive or transplantsurgery.

The treatment may involve assessing the subject (e.g., age, workingneeds of the subject, eye defect or disease, etc.), prescribing the useof moulding contact lenses, corneal inserts, corneal implants, orintrastromal rings to assist with the needed changes in the radius ofcurvature of the anterior surface of the cornea, and prescribing thecomposition described herein to be used in conjunction with the contactlenses or implants/inserts. The contact lenses or implants/inserts whichare prescribed and utilised by the subject can be used exert amechanical force on the cornea thereby inducing a change in shape, i.e.,the refractive power, of the cornea.

In certain preferred aspects, the cornea may be supported or shaped byuse of a corneal insert, corneal implant, or intrastromal ring inconjunction with the disclosed composition. Examples of commerciallyavailable devices include INTACS® and KeraRing intrastromal cornealrings. In another aspect, a moulding contact lens may be used inconjunction with the disclosed composition. The contact lens may be hardor rigid, or it may be a soft lens. Alternatively, the contact lens maycomprise both hard and soft portions. If a soft contact lens is used,more positive or negative curvature can be induce in the cornea, and thediscomfort in the subject's eyes will diminish as he or she adapts tothe contact lenses. If a hard contact lens is used, more mechanicalpressure can be exerted on the cornea. The contact lenses may be gaspermeable. Moulding contact lenses may be obtained from commercialsources. Examples of commercially available lenses include, at least,DreamLite, OK Lens, EyeDream, MiracLens, DreamLens, i-GO OVC, GOV, Wakeand See, CRT, Fargo/iSee, Emerald and Wave Contact Lens System lenses.

Once the contact lens, corneal insert, corneal implant, or intrastromalring is placed on/into the eye of the subject, the composition describedherein (e.g., eye drops) may be administered to the eye. In certaincircumstances, it may be desirable to pre-administer the compositionprior to placement of the contact lens, corneal insert, corneal implant,or intrastromal ring. Advantageously, the contact lenses, intrastromalrings, or other inserts/implants and the composition may be used inconjunction to produce a change in the shape, and thereby the refractivepower, of the cornea. The composition may be administered morefrequently to allow the cornea to adopt the desired change in shape. Incertain aspects, the composition is administered at least every 24, 12,or 8 hours. In other aspects, the composition is administered every 6hours. In certain other aspects, the composition is administeredapproximately every 3 hours. In yet other aspects, the composition isadministered approximately every 2 hours. In still other aspects, thecomposition is administered every hour.

Without wishing to be bound by any particular theory, the combined useof contact lenses, corneal inserts, corneal implants, or intrastromalrings and the composition described herein may induce changes in themolecular structure of the cornea and may induce changes in the cellsand proteins such as collagen (e.g., collagen type II) found in thecorneal stroma. The surface of the cornea is thereby made more uniform.By reducing irregularities in the surface of the cornea, the quality andclearness of all images (i.e., visual acuity) is improved.

For the calculation of the moulding contact lenses the flattestkeratometry is taken. One of skill in this art could also use thesteeper keratometry or an average of both and based on this cornealcurvature make the necessary calculations to flatten or steepen theradius of curvature of the anterior surface of the cornea and thuscorrect the refractive defect of the eye. The base curve of the mouldingcontact lens may be calculated based on the change in the refractivepower for each eye separately. In particular aspects, the base curve ofthe moulding contact lens may be calculated starting with one to fourflatter or steeper diopters, more preferably one to three flatter orsteeper diopters, even more preferably one to two flatter or steeperdiopters, depending on the refractive error that is required. Theperipheral base curve depends on the adaptation of the moulding contactlens and is calculated to be 0.5 mm of radius greater than the centralzone, but can vary depending on the design.

The diameter of the moulding contact lens used in accordance with theinvention may be from 8.0 mm to 18.0 mm. Commercially available lensesare produced with such diameters. In certain aspects, the mouldingcontact lens may be a hard contact lens with a diameter ranging from 8.0mm to 12.0 mm. In other aspects, the moulding contact lens may be a softcontact lens with a diameter ranging from 13.0 mm to 15.0 mm. Softcontact lenses may cover the entire cornea and go from sclera to sclera.In still other aspects, the moulding contact lens may be comprised ofhard and soft materials. The contact lens may be hard in the centre, outto approximately 12.0 mm, 13.0 mm, 14.0 mm, or 15.0 mm, and then soft inthe periphery out to 16.0 mm, 17.0 mm, and 18.0 mm. A larger contactlens, preferably a soft contact lens, may be used at night as a mouldingcontact lens.

The power of the moulding contact lenses can be determined to thenearest possible refractive power that the subject requires to seecomfortably. During the adaptation process with the moulding contactlenses, if the vision is not adequate for the needs of the subject, thesubject is prescribed eyeglasses while the subject is undergoingtreatment. As the cornea is being reshaped or has been reshaped, variousoptometric measurements may be repeated to confirm that the treatment isprogressing as planned and is adequate. Such measurements may includeassessment of visual acuity for near and far vision, orthotypes,keratometry measurements, objective and subjective retinoscopy, diagramsof the adaptation of the moulding contact lens, movement of the mouldingcontact lens, and comfort of the moulding contact lens.

After the measurements are taken, changes may be made to the treatmentprogram based on these measurements. With each evaluation, a decisionmay be made whether to continue with the same moulding contact lens orwhether a new contact lens should be used. In addition, the samedecision can be made with regard to the composition being used with themoulding contact lenses. Changes in the moulding contact lenses and/orin the composition can be made to induce the desired reshaping of thecornea over several weeks. In certain aspects, weekly periodic revisionsare performed during the first 8 weeks after beginning treatment.

The composition as described herein induces changes in the collagencontent of the cornea (e.g., collagen type II). Other aspects of theanatomy, histology, and physiology of the cornea may also be affected bycomposition. In certain aspects, the composition may be hypertonic orhypotonic to induce changes in corneal hydration. In other aspects, thecomposition may be used to change the molecular structure of the cornea(e.g., the extracellular matrix) and in this way augment or repair thecornea, or reshape the cornea to the desired curvature.

When reshaping the cornea, it may be desirable to co-administer one ormore enzymes to soften the cornea. Exemplary enzymes include but are notlimited to hyaluronidase, chondroitinase ABC, chondroitinase AC,keratanse, and stromelysin, which have been shown to work on variousproteoglycan components of the cornea. Included also are the enzymecollagenase, matrix metalloproteinase 1 (interstitial collagenase), andmatrix metalloproteinase 2 (gelatinase). Where the composition isco-administered with any such enzymes, it may be desirable to include avehicle such as a polymer (e.g., methylcellulose, polyvinyl alcohol,cellulose, etc.) in the composition to enhance the working of suchenzymes. Additional agents may be included to activate metalloproteinaseenzymes, e.g., interleukin-1α, tumour necrosis factor α/β and anysubtypes thereof, monosodium urate monohydrate, 4-amino phenylmercuricacetate, human serum amyloid A, human B₂ microglobin, and copperchloride. Also included may be carbamide (urea). Any combination ofthese agents may also be used.

The composition may also be co-administered with one or more enzymesthat degrade other sugars or proteins found in the cornea. Thecomposition may be co-administered with one or more anaesthetics used toreduce the irritation of the moulding contact lens or any cornealinsert/implant to the cornea. The composition may be co-administeredwith one or more lubricants to improve the comfort of the subject duringthe treatment. In other aspects, the composition may be co-administeredwith one or more anti-microbial agents such as anti-bacterial,anti-viral, and/or anti-fungal agents. The composition may also beco-administered with one or more vasoconstrictors. The person of skillin the art can determine the appropriate agents for co-administration tothe subject based on the condition being treated.

In certain aspects, the composition may be provided in a kit. The kitmay include one or more of: moulding contact lenses, lubricating eyedrops, cleaning or other solutions for the contact lenses, a contactlens carrying case, an extra pair of contact lenses, and instructionsfor wearing the contact lenses and using the composition. Thecomposition provided with the kit may be formulated to include the notedcombination of components (a TGFβ3 polypeptide (or variants or fragmentsthereof) plus dexamethasone (or derivatives thereof or related steroidalagents)), or the kit may include the components as separateformulations, to be mixed together prior to administration, or to beadministered together, i.e., by simultaneous or sequentialadministration.

EXAMPLES

The examples described herein are provided for the purpose ofillustrating specific embodiments and aspects of the invention and arenot intended to limit the invention in any way. Persons of ordinaryskill can utilise the disclosures and teachings herein to produce otherembodiments, aspects, and variations without undue experimentation. Allsuch embodiments, aspects, and variations are considered to be part ofthis invention.

Example 1: Overview of Experiments

In previous experiments, the inventors have shown that it is possible todirect keratocytes to differentiate down a neuronal lineage. Theexperiments described herein have aimed to investigate the potential ofkeratocytes to switch to a chondrocyte-like cells that secrete cartilagespecific collagen type II. This type of cartilage is thought to beexpressed during development (Linsenmayer et al. 1990). A further aimhas been to establish whether collagen type II deposition could beinduced in vivo in the corneas of live rats and whether this treatmentpositively affected the optical properties of the corneas. A stillfurther aim has been to determine whether keratocytes in keratoconictissue could be amenable to this method of cell reprogramming andsubsequent production of collagen type II rich ECM. Finally, theexperiments have aimed to evaluate the effect of type II collagendeposition on the biomechanical properties of the in vivo and ex vivotreated corneas using nanoindentation testing, a bioengineering approachthat enables analysis of hardness and elastic modulus.

Example 2: Tissue Samples Human Tissue

Cadaveric whole human corneas, keratoconic corneas obtained at the timeof transplant surgery, human limbal rims and surgeon cut DSEK caps(excess stromal tissue from Descemet's stripping endothelialkeratoplasty) were obtained from donors sourced through the New ZealandNational Eye Bank (Auckland, New Zealand). Human limbal rims werecollected after the central corneal button had been removed for cornealtransplantation surgery leaving a 2 mm corneal margin from the limbaljunction. Prior to the use of tissue, research ethics approval andconsent was obtained from the Northern X Regional Human EthicsCommittee. All tissue, until use, was stored in New Zealand Eye Bankmedium (2% FCS, 2 mM L-glutamine, 1×Anti-Anti in Eagles MEM) andtransported in New Zealand Eye Bank transport medium (Eye Bank mediumsupplemented with 5% dextran).

Animal Tissue

Ethics approval for animal studies was obtained from the University ofAuckland Animal Ethics Committee (application number R856). Eyes andcartilage from 6-8 week old adult male Wistar rats were obtained aftereuthanisation using a carbon dioxide chamber. The whole eye was removedfrom the animal and the cornea was carefully dissected out usingsurgical scissors with the aid of a dissecting microscope. The xiphoidprocess, which is part of the sternum that contains a thin, broad plateof cartilage at its end, was dissected out using a scalpel blade. Theanimal tissue was washed with povidone-iodine (PVP-I) and sodiumthiosulphate. The excess fat and tissue covering the cartilage wasscraped away with a blade. Freshly harvested eyes and cartilage werestored for a minimal amount of time in phosphate buffered salinesolution until use.

Example 3: Histological Analysis Tissue Preparation and Cryosectioning

Corneal and cartilage pieces (2 mm×2 mm) were embedded in OptimalCutting Temperature compound (OCT, Tissue-Tek, Sakura, The Netherlands)before being snap frozen in liquid nitrogen. Sections 10-15 μm thickwere cut using a Microm HM550 Cryostat (Thermo-Scientific, USA) andmounted on SuperFrost™ Plus electrostatic slides (Menzel-Glenser,Germany). Cryosections were stored at −20° C. until further use.

Cell and Tissue Culture

Tissue Digestion and Cell Preparation from Human and Rat Corneas

Limbal rims were dissected to isolate stroma from sclera in a class IIlaminar flow hood. Following this, the corneal epithelium andendothelium was gently scraped off with a keratome and discarded. DSEKcaps also received gentle scraping with a keratome to remove theepithelium. Remaining stromal tissue was then digested in 0.4% type IIcollagenase (Sigma-Aldrich), in Hanks balanced salt solution (GIBCO®,Life Technologies) at 37° C. with gentle mixing on an orbital shaker. Avariety of digestion times were used with 5 hours being the timerequired for optimal tissue digestion and cell viability.

After tissue digestion was complete the cells were pelleted bycentrifuging at 1200 rpm for seven minutes. The cells were thenresuspended in a minimal amount of an appropriate cell culture mediumand counted using a Leica DM IL bench top inverted microscope and aNeubauer hemocytometer. A 1:1 ratio of cell suspension added to trypanblue solution (0.04% trypan blue stock in PBS) was used with a minimumof three counts per sample and the average value taken.

Cell Culture of Corneal Keratocytes

All cell manipulations were performed in a class II laminar flow hoodusing aseptic technique. Isolated keratocytes were cultured in either 12or 24 well cluster plates (Falcon) on plastic or glass coverslips in 2-3ml of cell culture media. Cells were kept in a humidified incubator at37° C. with 5% CO₂. Culture media was changed after 24 hours then everytwo days subsequently or more frequently if required. Cultures wereviewed daily with a Leica DM IL bench top inverted microscope. For cellpellet culture, freshly obtained cells after tissue were pelleted bycentrifuging at 300 g for 7 minutes at 20° C. in a plastic conical tube.Appropriate culture media was added to the tubes. After 24 hours ofincubation at 37° C., the cells had contracted and formed a pellet whichdid not adhere to the walls of the tube. The pellets were cultured in 2ml of media in a humidified atmosphere of 5% CO₂ at 37° C. for threeweeks. Media was changed every other day.

Organotypic Slice Culture

Human and rat corneal and cartilage tissue was thin-sliced (1-2 mm) inan anteroposterior plane with a blade and the slices were placed in anorganotypic air-liquid interphase culture system (FIG. 2). Briefly, theexplants of healthy tissue were cultured on 0.4 μm pore size cellculture inserts (Millicell, France) at the interface between culturemedium and a CO₂ rich environment. Corneal sections were placedepithelium side up on cell culture plate inserts with 3 ml of culturemedium. The culture media was changed every other day.

Example 3: In Vitro Reprogramming Culture Media

Several custom made media were used as described in the table below.

TABLE 1 Cell culture media used Name Medium Base Other ComponentsFibroblast Dulbecco's 10% FBS, 1% Anti-anti proliferation Modified Eagle(100X stock), 1% medium Medium (DMEM) GlutaMAX ™ (100X stock) (LifeTechnologies, GIBCO ®) Chondrogenic Advanced DMEM 10 ng/ml TGFβ3 (Abcam,reprogramming (Life Technologies, ab52313), 10−⁷ M medium GIBCO ®)dexamethasone (Abcam, ab120743), 1% GlutaMAX ™ (Life Technologies,GIBCO ®) (100X stock), 1% Anti-Anti (100X stock) (Life Technologies,GIBCO ®) Control Dulbecco's 1% GlutaMAX ™ (100X stock), medium ModifiedEagle 1% Anti-Anti (100X stock) Medium (DMEM)

Chondrogenic Reprogramming of Keratocytes

Tissue slices were cultured in the chondrogenic differentiation mediumfor varying time intervals to determine the optimum time required forthe growth factor treatment. Samples were collected for each time point(Table 2). For obtaining a monolayer of cells, keratocytes were seededon glass coverslips at a density of 15×10⁴ per cm². The cells wereallowed to attach to the coverslips for 24 hours and culture media waschanged every other day. Cultures were maintained for up to 3 weeks.

TABLE 2 Experimental time points for corneal tissue slice culture Timepoint 1 Time point 1 Time point 1 Week 1 Week 2 Week 3 Sample 1chondrogenic control medium control medium differentiation medium Sample2 chondrogenic chondrogenic control medium differentiationdifferentiation medium medium Sample 3 chondrogenic chondrogenicchondrogenic differentiation differentiation differentiation mediummedium medium Sample 4 control medium control medium control medium

Example 4: In Vivo Reprogramming Gel Eye Drop Formulation for GrowthFactor Delivery

Eye drops were formulated using gellan gum which is a water solublepolysaccharide produced by the bacterium, Pseudomonas elodea, The use ofgel base formulation allows a prolonged corneal residence time andincreased ocular bioavailability of the therapeutic agent. Sincepolymeric gellan gum is an anionic polymer it undergoes in situ gellingin the presence of mono- and divalent cations such as Ca²⁺, Mg²⁺, K⁺,and Na⁺ (Bakliwal, & Pawar 2010). The electrolytes present in the tearfluid cause the gelation of the polymer when it is instilled in the eyeand this in turn results in a longer residence time and increasedbioavailability of the drug (Ludwig 2005). Based on previous formulationstudies, the polymer formulation is a non-irritant and safe for in vivouse (Rupenthal, Green, & Alany 2011).

A 0.5% solution was prepared by first heating distilled water to 80° C.followed by the addition of gellan gum (Kelcogel™ USA) with constantstirring. Once the powder was completely dissolved, the solution wascooled and stored at 4° C. The appropriate amounts of growth factorswere added to the runny gel with constant stirring. A ten times higherconcentration of growth factors than that used in the culture medium wasused to make up for the drug lost through naso-lacrymal drainage andblinking. The eye drop gel included a final concentration of 100 ng/mlTGFβ3 and approximately 4 μg/ml dexamethasone.

Treatment with Neurogenic and Chondrogenic Factors

The animals were manually restrained and approximately 15 μL of the eyedrops were instilled in the right eye (FIG. 3). The contra lateral eyewas used as the control eye. Thrice daily eye drops were administeredfor up to 5 days for neuronal specification and for up to 8 weeks forchondrogenic specification.

Example 5: Immunohistochemical (IHC) Analysis Tissue Harvesting andTreatment

At the end of the treatment the animals were euthanised using a carbondioxide chamber. The eyes were harvested and rinsed in phosphatebuffered saline. The corneas were then dissected out carefully and fixedin 4% paraformaldehyde (PFA) for 1 hour and treated with sucrosesolution in order to cryoprotect the tissue before freezing andsectioning. Sucrose as a cryoprotection is a dehydrant that prevents theformation of ice crystal artefact in frozen tissue sections. In the caseof slow freezing of the tissue cryoprotection is particularly important.

Briefly, the corneas were immersed in 20% sucrose solution for 5 hoursat 4° C. and then moved to a 30% sucrose solution and kept at 4° C.until the tissue sinks (usually overnight). The corneas were thenembedded in OCT compound and immersed in liquid nitrogen to bring aboutrapid freezing. The frozen blocks of tissue were stored at −80° C. untilfurther use. Approximately 10-15 μm thick cryostat sections were mountedon SuperFrost™ Plus slides and the slides were stored at −80° C. untilneeded. In the case of cell cultures, the cells cultured on coverslipswere rinsed with PBS and fixed with 4% PFA for 15 minutes. Coverslipswere immersed in PBS until further use.

Immunohistochemistry

For tissue cryosections, before carrying out immunohistochemistry, theslides were kept at room temperature for 15-20 minutes. The OCT waswashed off using PBS and the zone around the tissue demarcated using awax pen. The tissue slices were first incubated with a blocking solutionof 10% normal goat serum for 1 hour followed by overnight incubationwith the appropriate dilution of primary antibody at 4° C. The slideswere then rinsed three times in PBS before incubation with theappropriate dilution of secondary antibody. The secondary antibody wasleft on for 2 hours at room temperature. Slices were counterstained withthe nuclear marker 4′, 6′-diamidino-2-phenylindol (DAPI) and mounted inCitifluor antifade agent (ProSciTech, Australia). An Olympus FluoView™FV-1000 confocal laser scanning microscope (405 nm, 473 nm, and 559 nmwavelength lasers) and Leica DMRA fluorescence microscope were used forimaging.

TABLE 3 Antibodies used Dilution Antibody Supplier/Cat. No. used Primaryantibodies Mouse anti Collagen Type I Abcam/ab63080  1:2000 Mouse antiCollagen Type II Millipore/MAB8887 1:200 Mouse anti Collagen Type IIIBiogenesis/2150-0081 1:100 Mouse anti Vimentin Sigma/V6630  1:1000 Mouseanti α Smooth muscle Novocastra/NCL-SMA 1:100 actin Secondary antibodiesGoat anti mouse Alexa 568 Molecular probes ®/A-11031 1:500 Goat antirabbit Alexa 488 Molecular Probes ®/A-11034 1:500 Goat anti mouse Alexa488 Molecular Probes ®/A-11001 1:500

Example 6: Gene Expression Analysis

RNA Isolation and cDNA Synthesis

The mRNA extraction from samples was carried out using the PureLink® RNAMicroKit (Invitrogen). In brief, tissue samples were mixed with 0.75 mlTRIzol® and carrier RNA and homogenised using a hand held homogeniser(PRO Scientific, Inc.). The samples where then incubated with 0.2 ml ofchloroform followed by centrifugation at 12000 rpm and 4° C. for 15minutes. The upper phase was separated and was mixed with ethanol andthen transferred to the collection column tube.

The RNA was collected on the column by centrifuging at 12000 rpm for 1minute. The flow-through was discarded and the extracted RNA treatedwith deoxyribonuclease (DNAse). The column was washed several times withthe buffers provided and the RNA was finally dispersed in ribonuclease(RNAse) free water. The concentration was determined using a NanoDrop®(Thermo Scientific) and the mRNA was stored at −80° C.

The SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen™, LifeTechnologies) was used to prepare cDNA. Briefly, 100 ng of RNA wasincubated at 25° C. for 10 minutes with VILO™ Reaction Mix, SuperScript®Enzyme Mix, and RNAse free water. The samples were then incubated at 42°C. for 120 minutes followed by 85° C. incubation for 5 minutes. The cDNAwas stored at −20° C.

Quantitative PCR Using TaqMan® Gene Expression Assays

TaqMan® Gene Expression Assays for the genes of interest were obtained.In the PCR step, 10 μL of TaqMan® Universal Master Mix II was combinedwith 1 μl, of the assay, approximately 25 ng of cDNA and 9 μL water tomake up a volume of 20 μL. The tubes were vortexed and centrifugedbriefly to spin down the contents. Each cDNA sample was prepared intriplicate and pipetted into a 384 well plate. 20 μL of each reactionmixture was loaded into each well of a MicroAmp® Optical 384-WellReaction Plate (Applied Biosystems). The plate was then covered with aMicroAmp® Optical Adhesive Film (Applied Biosystems) and the plate wascentrifuged briefly to eliminate air bubbles. The plate was transferredto the 7900HT Fast Real-Time PCR System and was run using the followingthermal cycling parameters, 50° C. for 2 min, 95° C. for 10 minutesfollowed by 40 cycles of 95° C. at 15 sec and 60° C. at 1 minute.Results were analysed as described in the previous section.

TABLE 4 TaqMan ® gene assays used for QPCR Gene symbol Gene name AssayID Col1a1 (Rat) Collagen, type I, alpha 1 Rn01463848_m1 Col2a1 (Rat)Collagen, type II, alpha 1 Rn01637085_m1 Col2a1 (Rat) Collagen, type II,alpha 1 Rn01637087_m1 Pop4 (Rat) Ribonuclease P protein subunitRn02347225_m1 p29 (housekeeping gene) COL2A1 (Human) Collagen, type II,alpha 1 Hs00264051_m1 CDKN1A (Human) Cyclin-Dependent KinaseHs00355782_m1 Inhibitor 1 (housekeeping gene)

Example 7: Testing of Biomechanical and Optical Properties of CorneasFollowing In Situ Stromal ECM Protein Deposition Examination of AnteriorSegment (Frontal Structures) of the Rodent Eye

Corneal biomechanics have been shown to be relevant in the diagnosis andtreatment of various corneal diseases and provide insight into thestructure of the cornea and its relation to corneal physiologicalfunction. Corneas that have undergone treatment to bring about thedeposition of ECM protein also need to be tested for corneal opacity asreduced transparency would be undesirable.

The Phoenix Micron IV Rodent eye Imaging System (Phoenix Research Labs)was used to examine the corneas of treated rats. Rats were first sedatedusing an intra-peritoneal injection of ketamine and Domitor® (3:2). Theslit-lamp attachment of the Micron IV imaging system was used to examinethe layers of the cornea in detail and check corneal integrity andtransparency. Retinal imaging was also done to check cornealtransparency. Following imaging, the rats were administered Antisedan®(atipamezole) for reversal of the sedative.

Nanoindentation Measurements of In Vitro and In Vivo Treated Corneas

Nanoindentation provides mechanical measurements of materials ofinterest through the application of ultra-small forces perpendicular tothe sample plane of interest and measurement of the resultant sampleindentation (Dias & Ziebarth 2013). Nanoindentation has recently emergedas a powerful tool for measuring nano- and microscale mechanicalproperties in tissues and other biomaterials (Ebenstein & Pruitt 2006).The more recent advancement of in situ scanning probe microscopy (SPM)imaging, where the nanoindenter tip is simultaneously used as a 3Dimaging device combined with nanoindentation has enabled a new wave ofnovel materials research (Dickinson & Schirer 2009). Force,displacement, and time are recorded simultaneously while ananoindentation tip is pushed into the corneal tissue under a controlledload. The forces applied during nanoindentation can be as small as a fewnanoNewtons or as large as several Newtons enabling a range of sizescales to be studied. Nanoindentation tests are output as aload-displacement curve which can be analysed using well definedequations to calculate the mechanical properties relating to rigidity,integrity, and elasticity of the cornea.

Human keratoconic corneas were put into organotypic culture either incontrol medium or in medium containing the specific ECM protein inducingreprogramming factors. Nanoindentation measurements were then taken atthe end of the treatment time. For the in vivo study, the animals weremanually restrained and approximately 15 μL of the gel eye dropformulation containing the reprogramming factors were instilled in theright eye of each Wistar rat. The contra lateral eye was used as thecontrol eye. Eye drops were administered thrice daily for up to sevenweeks. Nanoindentation measurements were recorded after week 1, week 3,or week 7 of the treatment period on isolated eyes.

Nanoindentation testing was carried out at the Chemical and MaterialsEngineering lab at the University of Auckland. In order to test thecornea in its natural position, a mould was required fornanoindentation. Previous studies have used polystyrene and blue tack tohold the corneas in place. The effect of the mould deforming under theload was a potential source of error so a hard mould was decided on fortesting. The first material that was used to create a mould wasconventional play dough. This was formed to the exact shape andcurvature of human cornea samples (FIG. 6(A)). The play dough was thenleft to harden over the next two days before being used in testing. Thetesting of the rat eyes was slightly different as the entire globe wasused. To hold the globes in place a petri dish filled with a resin andhaving small indent to hold the globe was used (FIG. 6(B)). PBS was usedto keep the samples from drying out.

Because the samples are very soft biological samples a conosphericalfluid tip was used for all nanoindentation testing. The indent load usedfor the human samples was 50 μN. For the rat globes a range of loadsbetween 3 and 5 μN were used. The fibre optic light was switched on andthe sample placed directly under the stream of light from themicroscope. The central section of the cornea was placed directly in thestream of light as accurately as possible (FIG. 6(C)). The sample wasfocused by adjusting the Z slider until the surface of the cornea couldbe observed in good resolution. To ensure that the focus was on thecentral highest point of the cornea sample, the view was moved in the xand y directions to observe how the focus changed.

Once the data collection point was focused on the centre of the cornea,the sample boundary was defined and a quick approach was performed.Before indenting the load function had to be set up correctly. Theactual indentation process is automated by the Hysitron Triboindenter®(FIG. 6(D)). The pre-defined load was placed on the indenter tip whichpenetrates the sample until it reaches a defined limit. The tip was thenheld for 10 seconds before the tip was unloaded from the sample. Thehardness of the sample is determined by the area of residual indentation(Ar) after the tip is unloaded.

${Hardness} = \frac{{Maximum}\mspace{14mu} {Load}\mspace{14mu} (P)}{{Area}\mspace{14mu} {of}\mspace{14mu} {residual}\mspace{14mu} {indentation}\mspace{14mu} ({Ar})}$

Where P_(max) is the maximum indentation load and Area is the contactarea of the conospherical tip with the sample. The reduced elasticmodulus is a representation of the elastic modulus in both the sampleand the indenter tip as shown by the following equation:

$\left( \frac{1}{Er} \right) = {\left( \frac{1 - {vi}^{2}}{Ei} \right) + \left( \frac{1 - {vm}^{2}}{Em} \right)}$

Where i referrers to the indenter and m refers to the sample material.The reduced elastic modulus tells us how elastic a sample is. Becausethe same indenter tip is used for each test the reduced elastic moduluscan be used to compare the elasticity in each sample being tested.

Example 8: Adult Human Corneal Keratocytes Produce Cartilage SpecificCollagen Type II Upon Treatment with Exogenous TGFβ3 and Dexamethasone

It is known that one growth factor may act on several types of cellswith similar or varied effects whilst more than one growth factor mayshare similar biological functions. When choosing growth factors,cytokines, and chemicals that might bring about collagen deposition inthe corneal stroma it was important to consider the known effects ofcertain exogenous factors. In the present experiments, a combinationtreatment of TGFβ3 and dexamethasone was utilised.

Most of the evidence for the effects of TGFβ3 and dexamethasone has beenobtained by studies done on their effects on stem/progenitor cells(Schuldiner, Yanuka, Itskovitz-Eldor, Melton, & Benvenisty 2000;Worster, Nixon, Brower-Toland, & Williams 2000). A combination of TGFβ3and dexamethasone has been previously used to induce progenitor cells todifferentiate into chondrocytes in vitro (Diekman, Rowland, Lennon,Caplan, & Guilak 2009; Johnstone et al. 1998; Kolambkar, Peister, Soker,Atala, & Guldberg 2007; Winter et al. 2003). Furthermore, dexamethasone,a synthetic steroid drug has been used to treat inflammatory eyeconditions. Therefore a combination of TGFβ3 and dexamethasone was usedin the chondrogenic differentiation medium to drive the differentiationof keratocytes towards a chondrocyte phenotype.

In the present experiments, the expression of type I and type IIcollagen was specifically noted. It is known that fibrillar types ofcollagen such as types I and II self-assemble and crosslink to formhighly crystalline fibres exhibit a very high stiffness, lowextensibility and a remarkable elastic energy storage capacity (Wells2003). It is the crosslinking which contributes towards the stiffnessand tensile strength of the fibres.

The corneal stromal extracellular matrix (ECM) is composed of tightlypacked heterotypic collagen fibrils made up mostly of collagen types Iand V. Similar to corneal fibrils, cartilage fibrils are heterotypic(made up of types II and XI) and have a uniform diameter of 25 nm(slightly smaller than corneal fibrils) (Mendler, Eich-Bender, Vaughan,Winterhalter, & Bruckner 1989). Collagen II is the major fibrilcomponent of cartilage and is similar to collagen I in that the moleculeessentially consists of a single uninterrupted helical domain 300 nm inlength. Owing to their similarities, collagens II and XI are consideredto be the cartilage analogues of collagens I and V (corneal stromacollagens) in other tissues.

In the present experiments, corneal keratocytes from adult corneas wereseeded in either the chondrogenic differentiation medium containingTGFβ3 and dexamethasone or a standard fibroblast proliferation medium.Within 2-3 days the keratocytes seeded in the chondrogenicdifferentiation media formed cell aggregations/spheres (FIG. 7(A))approximately 50-100 μm in diameter. The spheres labelled for thechondrocyte specific collagen type II in the central portion and nestinaround the periphery (FIG. 7(B)). Furthermore, once the spheres wereplaced in the fibroblast proliferation media cells from the spheresstarted spreading outwards (FIG. 7(C)) to populate the culture dishthereby forming a cell monolayer. The regions where the cells had oncebeen aggregated labelled for collagen type II whereas the cells inmonolayer did not (FIG. 7(D)).

Keratocytes seeded in the fibroblast proliferating medium formed an evenmonolayer of fibroblast-like cells (FIG. 8(A)) which did not label foreither nestin or collagen type II (FIG. 8(B)). When the media waschanged to chondrogenic differentiation medium there were no changes inthe appearance of the culture and cells remained collagen type IInegative. These results suggest that cell aggregation appears to beimportant for cartilage-like ECM production. Keratocytes seeded intofibroblast proliferation medium failed to form the necessary cellaggregations. Therefore, in order to form fibroblast clusters, theconfluent fibroblasts were dissociated from the culture dish, pelleted,and grown as a pellet culture in chondrogenic differentiation medium fora further three weeks. Cell pellets labelled positive for the cornealstroma specific ECM protein keratocan but not the cartilage specific ECMprotein type II collagen (FIGS. 8(F) and (G)).

Example 9: Keratocytes in Adult Human Corneas and Adult Rat CorneasSecrete Collagen Type II Containing ECM when Treated with TGFβ3 andDexamethasone

Slices of adult human cornea were placed in organotypic slice culture ineither control medium or chondrogenic differentiation medium for twoweeks. The tissue slices were then labelled for the chondrocyte specificECM protein collagen type II and the native corneal collagen type I.Positive labelling was seen only in the TGFβ3 and dexamethasone treatedcorneas (FIGS. 9(C) and 10(B)). It was found that a treatment period oftwo weeks resulted in deposition of type II collagen within the stromalECM of treated corneas (FIG. 9(C)). Treatment for 1 week did not resultin any visible deposition of type II collagen in the stromal ECM (FIG.9(B)).

The amount and pattern of the native collagen type I appeared to beslightly altered in the treated corneas. In general, the intensity ofthe labelling was similar but the distribution was more extensive andthe amount of labelling was higher in the untreated corneas (FIG. 9(D)).Furthermore, the newly produced type II collagen was laid evenly and inan ordered fashion in the ECM without forming any large masses oraggregates. The labelling was clearly seen along the pre-existingcollagen framework of the corneal stroma and was distributed across theentire thickness of the stromal layer.

The in vitro human corneal tissue experiment was then extended to an invivo rodent study wherein the right corneas of male Wistar rats weretreated for two weeks with a thrice daily administration of 15 μl of agellan gum based eye drop formulation of TGFβ3 and dexamethasone. Aftertwo weeks the rats were euthanised and the corneas processed forimmunohistochemistry. Only the treated corneas labelled positive forcollagen type II with a higher degree of deposition observed in theanterior part of the cornea (FIGS. 10(D) and (E)). Thus, only cornealslices cultured in the chondrogenic differentiation medium were positivefor type II collagen. Furthermore, type II collagen was laid down inuniform layers along the pre-existing collagen framework of the stroma.

Example 10: Induction of Collagen Type II Deposition in KeratoconicCorneas

The inventors next looked to confirm that the in vivo reprogrammingobserved in their studies could be utilised in treatments forkeratoconus. Experiments were carried out to affirm that keratocytes inkeratoconic corneas were amenable to the induction of collagen type IIdeposition. Keratoconic corneal buttons obtained after cornealtransplant surgery were placed into culture as soon as they wereobtained. Half of each button was put into control medium and the otherhalf placed in chondrogenic differentiation medium and maintained for 2weeks. After 2 weeks the tissue was processed for eitherimmunohistochemistry or mRNA extraction. The stromal ECM of only thetreated half of the cornea was positive for type II collagen (FIG.11(B)). Although the intensity of the labelling was lower in keratoconictissue when compared to normal corneal tissue, the labelling pattern wassimilar and followed an ordered arrangement along the backbone ofpre-existing collagen lamellae.

Vimentin labelling revealed stark differences between keratocytes in theuntreated and treated keratoconic corneas. In general the keratocytedensity was lower in the untreated corneas with a scarcity of cells inthe posterior part of the cornea (FIG. 11(C)). Also, the keratocytes intreated corneas appeared more filamentous and complete in morphologywhen compared to keratocytes in untreated corneas (FIGS. 11(E) and (F)).Keratocytes in treated corneas were longer and had a larger number ofcell processes which labelled strongly for Vimentin when compared to thekeratocytes in the untreated corneas.

Example 11: TGFβ3 and Dexamethasone Treatment does not Induce Depositionof Fibrotic Proteins or Cause Corneal Opacity

Human corneas cultured in the chondrogenic differentiation medium for upto three weeks were labelled for collagen type III and αSMA which areassociated with fibrosis and scarring (Gabbiani 2003; Karamichos et al.2012). There was no evidence of any fibrotic matrix deposition, on theother hand there was a higher degree of αSMA labelling in the controltissue (FIG. 12). These results confirm previous findings that, unlikeTGFβ1 and TGFβ2, TGFβ3 does not induce the differentiation of cornealkeratocytes into myofibroblasts.

Slit lamp examination was performed on the live rats throughout thestudy period. Upon examination, treated and untreated corneas wereindistinguishable with no signs of scarring or opacity. Back of the eyeimaging to reveal the blood vessels showed clear corneas which did notobstruct the passage of light (FIGS. 13(A) and (B)) and in vivo crosssection imaging of the rat cornea using the Micron IV lens revealedtransparent corneas through which light easily passed (FIGS. 13(C) and(D)). There was no sign of any corneal opacity or cloudiness which wouldlead to the obstruction of light passing through the cornea.

Example 12: Change in mRNA Expression of Collagen Type II and Type IUpon Treatment In Vivo

Rat corneas which were treated in vivo for 1 week, 7 weeks, and 3 weeksfollowed by a non-treatment period of 4 weeks were subjected toquantitative gene expression analysis. The aim was to determine whethertype II collagen expression decreases again and/or permanently ceasesafter growth factor treatment is withdrawn. The effect of the treatmenton native corneal collagen type II was also investigated.

When compared to the 7 weeks treated corneas, the 1 week treated corneasexpressed very high levels of type II collagen. The expression levelsdropped considerably upon withdrawal of the treatment as indicated bythe first graph in FIG. 35. For type I collagen expression, the 1 weekand 7 week treated corneas were each compared to their untreatedcorneas. It was found that there was an initial spike in type I Collagenexpression after 1 week treatment but by week 7 type I Collagenexpression was significantly lower and comparable to its expression inthe untreated cornea (FIG. 14 (B)).

Example 13: Change in Biomechanical Properties of In Vitro and In VivoTreated Corneas

It was hypothesised that the laying down of type II collagen wouldaffect the stiffness and elasticity of the corneas. In order to evaluatethese changes, the in vivo rat corneas and ex vivo treated human corneasand their matching controls were subjected to nanoindentation testing.

When compared to the untreated controls the 1 week in vivo treated ratcorneas did not have a significant increase in either hardness orelasticity (FIG. 15). In the 3 week in vivo treated corneas, there was aclear difference between the treated and control eye. Each of thecorneas was tested up to eight times and the resulting load deformationgraphs obtained showed good reproducibility (FIG. 16). In the right eyeexposed to the growth factor treatment, both the hardness and reducedelastic modulus were markedly higher. A matched pair of keratoglobuscorneas that were cultured ex vivo in either the control medium or thechondrogenic differentiation medium for 6 weeks were also subjected tothe same biomechanical testing. Once again, testing revealed asignificant increase in hardness and elastic modulus in the treatedcornea (FIG. 17).

Example 14: Comparative Combinations of Growth Factors and Steroids

An ex vivo study on sheep corneas was carried out in order toinvestigate the efficacy of other growth factor-steroid combinations inchondrogenic differentiation of corneal keratocytes.

Fresh sheep eyes were obtained from Auckland Meat Processors. Thecorneas were immediately excised and washed with povidone-iodine (PVP-I)and sodium thiosulphate solution. Then, 8 mm discs of sheep cornealtissue were cut using a trephine. One sheep corneal disc was placed ineach of the culture conditions (outlined in Table 5) for 3 weeks. Thecorneal discs were then placed in an organotypic air-liquid interphaseculture system.

Briefly, the explants of healthy tissue were cultured on 0.4 μm poresize cell culture inserts (Millicell, France) at the interface betweenculture medium and a CO₂ rich environment. Corneal sections were placedepithelium side up on cell culture plate inserts with 3 ml of culturemedium. The culture media was changed every other day. The basal mediumused was Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1%Anti-Anti (antibiotic-antimycotic solution) and 1% GlutaMAX™ (GIBCO®).At the end of 3 weeks, each corneal disc was fixed in 4%paraformaldehyde (PFA) for 1 hour and treated with sucrose solution inorder to cryoprotect the tissue before freezing and sectioning.

In brief, the corneas were immersed in 20% sucrose solution for 5 hoursat 4° C. and then moved to a 30% sucrose solution and kept at 4° C.until the tissue sank (usually overnight). The corneas were thenembedded in OCT (optimal cutting temperature) compound and immersed inliquid nitrogen to bring about rapid freezing. The frozen blocks oftissue were stored at −80° C. until further use. Approximately 4-6 40 μmthick cryostat sections were mounted on SuperFrost™ Plus slides and theslides were stored at −80° C. until needed. The corneal sections werethen labelled for collagen type II.

For immunohistochemistry, the slides were kept at room temperature for15-20 minutes. The OCT was washed off using PBS and the zone around thetissue was demarcated using a wax pen. The tissue slices were firstincubated with a blocking solution of 10% normal goat serum for 1 hrfollowed by overnight incubation with mouse anti collagen II antibody(Millipore/MAB8887) at 4° C. The slides were then rinsed three times inPBS before incubation with the appropriate dilution of goat anti mouseAlexa Fluor® 488 secondary antibody (Molecular Probes®/A-11001). Thesecondary antibody was left on for 2 hours at room temperature. Sliceswere counterstained with the nuclear marker 4′,6′-diamidino-2-phenylindol (DAPI) and mounted in Citifluor antifadeagent (ProSciTech, Australia). An Olympus FluoView™ FV-1000 confocallaser scanning microscope (405 nm, 473 nm and 559 nm wavelength lasers)and Leica DMRA fluorescence microscope were used to visualise labelling.

Table 5 depicts the findings from this study. FIG. 20 showsrepresentative images of collagen type II labelling in corneal sections,in each of the conditions.

TABLE 5 Tested combinations of growth factors and steroids Collagen typeII deposition Representative Growth factor-steroid combination (Y/N)image BMP6 N FIG. 20A BMP6 + hydrocortisone N FIG. 20B TGFβ₃ +hydrocortisone N FIG. 20C BMP6 + dexamethasone N FIG. 20D TGFβ₃ +prednisone N FIG. 20E TGFβ₃ + Triesense ® N FIG. 20F TGFβ₃ +dexamethasone Y FIG. 20G-H

The results confirmed that the combination of TGFβ3 and dexamethasone isthe only tested combination that elicited the desired response from thetarget cells (FIG. 20, panels G-H). The other growth factor-steroidcombinations failed to produce the desired changes in collagen type IIin keratocytes (FIG. 20, panels A-F). The results also confirmed thereprogramming of keratocytes in sheep corneas (FIG. 20, panels G-H).

Previous studies have shown other growth factors and other steroidcompounds to be unsuitable for corneal treatment and repair. TGFβ1 andTGFβ2 both produce fibrotic scarring (Carrington, Albon et al. 2006;Desmouliere, Chaponnier et al. 2005; Jester, Huang et al. 2002; Cowin etal. 2001; Shah et al. 1995). EGF negatively regulates chondrogenesis(Yoon 2000). Estrogen also negatively regulates chondrogenesis (Kato &Gospodarowicz 1985). Hydrocortisone has been shown to promote adipogenicrather than chondrogenic differentiation (Ghoniem et al. 2015; Lee, Kuoet al. 2004). These earlier studies show the significance of the presentfindings on TGFβ3 and dexamethasone, which act together to promotechondrogenic differentiation of corneal keratocytes and scar freecorneal healing.

Example 15: Comparative Dosages for TGFβ3 and Dexamethasone

Prior to an in vivo study, experiments were performed to identify thevarious effective dosages for ex vivo treatments. A dose range study wascarried out for TGFβ3 and dexamethasone by culturing sheep corneas inculture media containing these two factors in varying concentrations.

Fresh sheep eyes were obtained and corneas were excised and treated asnoted in Example 14. One sheep corneal disc was placed in each of the 16culture conditions (FIG. 21) for 3 weeks. The corneal discs werecultured and then subjected to immunohistochemical and microscopicanalysis as noted in Example 14. FIG. 21 shows representative images ofcollagen type II labelling in corneal sections in each of theconditions.

This study revealed that lower concentrations of TGFβ3 (2-4 ng/mL) anddexamethasone (1-10 nM) had lower efficacy ex vivo (FIG. 21, first andsecond rows). Higher doses, i.e., 8-10 ng/mL TGFβ3 and 100-1000 nMdexamethasone were efficient in inducing collagen type II deposition(FIG. 21, third and fourth rows).

These results confirmed the use of 100 nM dexamethasone and 10 ng/mLTGFβ3 as effective concentrations (FIG. 21, third row). Higherconcentrations of dexamethasone (1000 nM, i.e., 400 ng/mL) were alsoshown to be effective (FIG. 21, fourth row). It was noted that thedexamethasone concentrations tested in this study were considerablylower than the concentrations used in commercially available eye drops(i.e., 1 mg/mL dexamethasone).

Example 16: Overview of Experimental Observations and Results

A combination of TGFβ1 and dexamethasone has been previously used toinduce progenitor cells to differentiate into chondrocytes in vitro(Diekman et al. 2009; Johnstone et al. 1998; Kolambkar et al. 2007;Winter et al. 2003). In other studies, a side population of cornealstromal cells has been shown to produce a matrix made up of thecartilage specific collagen II under similar chondrogenicdifferentiation conditions (Du, Funderburgh, Mann, SundarRaj, &Funderburgh 2005). It has also been reported that scleral cells afterfour weeks in a chondrogenic differentiation medium containing TGFβ1 andBMP2 expressed cartilage specific markers including aggrecan, andcollagen type II. Furthermore, human scleral cells have been shown toretain their chondrogenic potential in vivo after being transplantedinto a rat cartilage defect (Seko et al. 2008). It is known that thefibroblastic cells of the sclera and the corneal stroma share a commonembryological origin.

As shown herein, keratocytes seeded in culture medium containing TGFβ3and dexamethasone and in the absence of serum spontaneously formed cellspheroids within 2-3 days by cell aggregation and by three weeks thesecell clusters labelled positive for cartilage specific type II collagen.Initially upon treatment with TGFβ3 and dexamethasone, type I collagenexpression was also increased. When the medium was changed to a controlmedium containing fetal calf serum the cell clusters dispersed into amonolayer of cells. Cells growing in the monolayer no longer expressedtype II collagen. These results suggest that cell aggregation orenvironment might be important in collagen type II induction.

Notably, keratocytes which were first proliferated as fibroblasts inserum containing medium did not secrete collagen type II when the mediumwas changed to the TGFβ3 and dexamethasone containing chondrogenicdifferentiation medium. This suggests that once proliferated asfibroblasts the cells lose the ability to differentiate along achondrogenic pathway. Further to this, fibroblasts grown inthree-dimensional culture in chondrogenic differentiation medium as apellet also failed to express cartilage specific collagen type II. Theseresults suggest that the quiescent keratocyte phenotype and cellaggregation are important to chondrogenic differentiation.

It is shown herein that ex vivo culture of normal and keratoconiccorneas in chondrogenic differentiation media revealed uniformdeposition of type II collagen along the stromal lamellae. Everykeratocyte within the corneal stroma was associated with the collagentype II labelling, once again suggesting that the reprogramming into achondrogenic phenotype is stochastic and confirming that resultsobtained from the in vitro cell culture were not as a result ofproliferation of a side population of progenitor cells. Furthermore, invivo treatment of corneas in rats also caused the deposition of type IIcollagen in a manner similar to that seen in ex vivo culture. However,stronger immunolabeling of type II collagen was seen in the anteriorpart of the cornea when treated in vivo, most probably reflecting easierdiffusion of growth factors into the anterior layers of the stroma fromthe ocular surface.

Studies looking at differences in keratocyte density in keratoconiccorneas have reported an overall decrease in cell density. The resultshere also confirm this. However, unlike other studies which havereported a marked decrease in cell density in the anterior part of thestroma (Hollingsworth, Efron, & Tullo 2005; Ku et al. 2008; Mencucci etal. 2010; Niederer et al. 2008), the results here indicate a markeddecrease in keratocyte density in the posterior part of the stroma ofthe untreated keratoconic cornea also. In keratoconus there is a generalthinning of the cornea. It is not known, however, whether this is due tothe apoptosis of keratocytes and subsequent decreased production of ECMor whether keratocyte apoptosis is secondary to the process of cornealthinning.

As shown herein, the treated half of the keratoconic cornea which wascultured in the chondrogenic medium containing TGFβ3 and dexamethasonehad an increased keratocyte density when compared to the control.Furthermore, the posterior region of the stroma appeared to berepopulated by keratocytes. The keratocytes in the treated half alsoappeared to look healthier with large prominent nuclei and several cellprocesses. This indicates that the treatment with the two factors havepossibly caused keratocytes to proliferate and repopulate the stroma, inparticular the posterior part which was devoid of keratocytes.

Collagen crosslinking, one of the current treatments for keratoconus,results in an initial period of keratocyte apoptosis in the anteriorpart of the stroma. This is then followed by a period of repopulation ofthe stroma by the keratocytes. Keratocyte cell death is generally seenin response to an injury and in the case of crosslinking is understoodto be as a result of UVA-induced cellular damage. This apoptoticresponse is thought to have evolved in order to protect the cornea fromfurther inflammation (Wilson, Netto, & Ambrosio 2003).

Stromal haze which can last up to several months is also observed afterthe crosslinking treatment. It has been attributed to the increase incollagen diameter and spacing between the collagen fibrils which resultsin the modification of the corneal microstructure. Most studies havereported a decrease in corneal haze between 6-12 months after thetreatment (Greenstein, Fry, Bhatt, & Hersh 2010; Mazzotta et al. 2008).Although there have been several clinical observations of the corneascarried out after the crosslinking treatment there is ambiguityregarding the cause of the corneal haze and other possible downstreameffects of the treatment. The fact that it takes several months forcorneas to be repopulated and become clear suggests that thecrosslinking might be triggering a wound healing response within thestroma.

In this study, even upon long term (up to 8 weeks) in vitro and in vivotreatment there was no evidence of corneal opacity. This is probably dueto the deposition of the collagen II in uniform layers along thepre-existing collagen lamellae. Deposition of collagen type III(associated with fibrosis) and alpha-smooth muscle actin (duringmyofibroblast formation) leads to opacity and scarring. Both these areseen during corneal wounding. Neither of these proteins was expressed inthe treated corneas suggesting that wound healing cascades which couldbring about scarring were not being triggered.

As described herein, quantitative measurement of type II collagen mRNAexpression showed that its expression was significantly lowered uponwithdrawal of TGFβ3 and dexamethasone. This suggests that thereprogramming of keratocytes is not irreversible and the subsequentdeposition of type II collagen in the ECM can potentially be controlled.This is important for the development of therapeutic methods, as itwould not be desirable to induce irrepressible ECM deposition.

Nanoindentation has been employed in the assessment of postoperativetherapeutic methods such as crosslinking for keratoconus (a cornealdystrophy) and post-LASIK ectasia in the eye. In one study done on humancadaver corneas it was found that collagen crosslinking caused atwo-fold increase in the elastic modulus in the anterior corneal stromawhile the posterior stroma was unaffected by the treatment (Dias,Diakonis, Kankariya, Yoo, & Ziebarth 2013). In this study, anteriorcorneal elasticity was measured. In addition, the results in this studydo indicate that posterior stroma keratocyte density was altered in theTGFβ3 and dexamethasone treated corneas.

While nanoindentation does not measure the properties of the individualcollagen fibrils it can measure the changes in the inherent elasticproperty of the cornea which will be altered on collagen II depositionwith a subsequent increase in collagen crosslinking. Structuraldifferences within the stroma are reflected in the correspondingdifferences in biomechanical properties. The results here show thatthere was almost a three-fold increase in elastic modulus and hardnessin the growth factor treated rat corneas. These results indicate thatthe treatment results in a stiffer cornea with higher elasticity. Theelastic modulus is a measure of a substance's resistance to beingdeformed elastically and therefore a higher elastic modulus indicatesthat a material is more difficult to deform. In this study, asignificant increase in hardness and elastic modulus in 3 week treatedcorneas when compared to 1 week treated corneas is consistent with theimmunohistochemical labelling results that show at least 2-3 weeks oftreatment is required for the laying down of detectable layers of typeII collagen.

The immunohistochemical labelling results coupled with the geneexpression studies and biomechanical testing show that keratocyteswithin an intact cornea are amenable to reprogramming along achondrogenic pathway by treatment with TGFβ3 and dexamethasone. Thereprogramming by combined TGFβ3 and dexamethasone treatment isstochastic and may be controlled via the modulation of the growth factortreatment period to result in stiffer, more elastic corneas. Notably,administration of both agents is required; when TGFβ3 and dexamethasoneare tested separately, no collagen type II production in keratocytes isobserved. A novel treatment is therefore proposed for keratoconus andother eye conditions using in vivo tissue engineering, by administrationof TGFβ3 and dexamethasone, as described herein.

Example 17: Large Animal Model to Investigate Reshaping of the CorneaReshaping the Cornea Whilst Delivering the Optimal Regimen in a SheepModel

Additional experiments are carried out to use a large animal model todemonstrate reshaping of the cornea. For these experiments, a largeanimal model is used to allow placement of prescription contact lenses.Sheep are used as a model animal, as their eyes are comparable in sizeand physiology to that of humans. In addition, housing facilities areavailable at Lincoln University, Christchurch. It is noted also thatsheep have a mild temperament, and are amenable to handling.

Sheep are sedated in accordance with standard operating procedure in thehousing facility. The eye drop formulation with optimal TGFβ3 anddexamethasone concentrations (volume scaled) based upon the rodent doseoptimisation studies are instilled in the right eye followed by theplacing of corneal INTACS® (or similar scleral rings) to hold thedesired curvature of the cornea during collagen deposition (FIG. 18).Eye drops are continued to be administered either once or twice daily(as determined in rodent optimisation studies) for a period of threeweeks. The INTACS® are then removed and the animals are continued to behoused for a further three weeks or six months.

Before treatment and at the end of the treatment (when the INTACS® areremoved), corneal thickness and curvature measurements are taken. Theportable corneal pachymeter is used to detect changes in cornealthickness of treated versus control contralateral corneas in vivo. Aportable Pentacam® is used to measure corneal curvature as well ascorneal thickness of the sheep eyes before and after treatment (FIGS.19(E) and (F)). Corneal measurements are repeated again at three weeksafter lenses removal with the final (most accurate) Pentacam®measurements. These are taken after killing the animal but prior to eyeremoval for immunohistological and biomechanical analysis as describedabove for rodent corneas. In the unlikely event that the sheep areunable to tolerate a hard contact lens (signs of infection, inflammationor irritability), the study is continued without lenses, which allowscompletion of key parameters such as type II collagen deposition anddistribution, and biomechanical properties.

In view of the results, it is proposed to use in vivo tissue engineeringas described in detail herein, in combination with of a rigid gaspermeable OrthoK contact lenses (or similar) to permanently reshape andstabilise the cornea, providing treatment for common corneal defects,including myopia.

REFERENCES

-   Ashwin, P. T., & McDonnell, P. J. (2010). Collagen cross-linkage: a    comprehensive review and directions for future research. British    Journal of Ophthalmology, 94(8), 965-970.-   Cosar, C. B. et al. (2002). Indications for penetrating keratoplasty    and associated procedures, 1996-2000. Cornea, 21(2), 148-151.-   Cowin, A. J., Holmes, T. M., Brosnan, P., & Ferguson, M. W. (2001).    Expression of TGF-beta and its receptors in murine fetal and adult    dermal wounds. European Journal of Dermatology, 11(5), 424-31.-   Denniston A. K. O., Murray P. I. (2009) Oxford Handbook of    Ophthalmology (OUP). Second edition. Oxford: New York. Oxford    University Press.-   Desmouliere, A., et al. (2005). Tissue repair, contraction, and the    myofibroblast. Wound Repair and Regeneration, 13(1), 7-12.-   Dias, J., Diakonis, V. F., Kankariya, V. P., Yoo, S. H., &    Ziebarth, N. M. (2013). Anterior and posterior corneal stroma    elasticity after corneal collagen crosslinking treatment.    Experimental Eye Research, 116, 58-62.-   Dias, J. M., & Ziebarth, N. M. (2013). Anterior and posterior    corneal stroma elasticity assessed using nanoindentation.    Experimental Eye Research, 115, 41-46.-   Dickinson, M. E., & Schirer, J. P. (2009). Probing more than the    surface. Materials Today, 12(7), 46-50.-   Diekman, B. O., Rowland, C. R., Lennon, D. P., Caplan, A. I., &    Guilak, F. (2009). Chondrogenesis of adult stem cells from adipose    tissue and bone marrow: induction by growth factors and    cartilage-derived matrix. Tissue engineering Part A, 16(2), 523-533.-   Dobbins, K. R., F. W. Price Jr., W. E. Whitson. (2000). Trends in    the indications for penetrating keratoplasty in the Midwestern    United States. Cornea, 19(6), 813-816.-   Ebenstein, D. M., & Pruitt, L. A. (2006). Nanoindentation of    biological materials. Nano Today, 1(3), 26-33.-   Edmund, C. (1988). Corneal elasticity and ocular rigidity in normal    and keratoconic eyes. Acta Ophthalmologica, 66(2), 134-140.-   Edwards, M. et al. (2002). Indications for corneal transplantation    in New Zealand: 1991-1999. Cornea, 21(2), 152-155.-   Farquharson, C., Berry, J. L., Barbara Mawer, E., Seawright, E., &    Whitehead, C. C. (1998). Ascorbic acid-induced chondrocyte terminal    differentiation: the role of the extracellular matrix and 1,    25-dihydroxyvitamin D. European Journal of Cell Biology, 76(2),    110-118.-   Fredrick, D. R. (2002). Myopia. BMJ: British Medical Journal,    324(7347), 1195.-   Fukuchi, T., Yue, B., Sugar, J., & Lam, S. (1994). Lysosomal enzyme    activities in conjunctival tissues of patients with keratoconus.    Archives of Ophthalmology, 112(10), 1368.-   Funderburgh, J. L. (2000). Corneal proteoglycans. In: Proteoglycans:    Structure, Biology and Molecular Interactions, R. V. Lozzo, Editor.    Marcel Dekker.-   Funderburgh, J. L., Mann, M. M., Funderburgh, M. L., Corpuz, L., &    Roth, M. R. (2001). Proteoglycan expression during transforming    growth factor-induced keratocyte-myofibroblast transdifferentiation.    Journal of Biological Chemistry, 276(47), 44173.-   Funderburgh, J. L., M. M. Mann, and M. L. Funderburgh (2003)    Keratocyte phenotype mediates proteoglycan structure. Journal of    Biological Chemistry, 278(46): 45629.-   Gabbiani, G. (2003). The myofibroblast in wound healing and    fibrocontractive diseases. The Journal of Pathology, 200(4),    500-503.-   Ghoniem, A. A., Açil, Y., Wiltfang, J., & Gierloff, M. (2015).    Improved adipogenic in vitro differentiation: comparison of    different adipogenic cell culture media on human fat and bone stroma    cells for fat tissue engineering. Anatomy & Cell Biology, 48(2),    85-94.-   Greene, C. A. et al. (2013). Cells from the adult corneal stroma can    be reprogrammed to a neuron-like cell using exogenous growth    factors. Experimental Cell Research, 322(1), 122-132.-   Greenstein, S. A., Fry, K. L., Bhatt, J., & Hersh, P. S. (2010).    Natural history of corneal haze after collagen crosslinking for    keratoconus and corneal ectasia: Scheimpflug and biomicroscopic    analysis. Journal of Cataract & Refractive Surgery, 36(12),    2105-2114.-   Gurdon, J. B., D. A. Melton. (2008). Nuclear reprogramming in cells.    Science, 322, 1811-1815.-   Gordon, M. K., R. A. Hahn (2010). Collagens. Cell and Tissue    Research, 339(1), 247-257.-   Håkelien, A. M., P. Collas. (2002). Novel approaches to    transdifferentiation. Cloning & Stem Cells, 4(4), 379-387.-   Heng, B. C., Cao, T., & Lee, E. H. (2004). Directing stem cell    differentiation into the chondrogenic lineage in vitro. Stem Cells,    22(7), 1152-1167.-   Hollingsworth, J. G., Efron, N., & Tullo, A. B. (2005). In vivo    corneal confocal microscopy in keratoconus. Ophthalmic and    Physiological Optics, 25(3), 254-260.-   Ignotz, R. A. & Massagué, J. (1986). Transforming growth factor-beta    stimulates the expression of fibronectin and collagen and their    incorporation into the extracellular matrix. Journal of Biological    Chemistry, 261, 4337-4345.-   Ignotz, R. A., Endo, T., & Massague, J. (1987). Regulation of    fibronectin and type I collagen mRNA levels by transforming growth    factor-beta. Journal of Biological Chemistry, 262(14), 6443-6446.-   Jackson T. L. (2008) Moorfields Manual of Ophthalmology, Mosby,    Elsevier.-   Jester, J. V., Rodrigues, M. M., & Herman, I. M. (1987).    Characterization of avascular corneal wound healing fibroblasts. New    insights into the myofibroblast. The American Journal of Pathology,    127(1), 140.-   Jester, J. V., et al. (2002). TGFβ induced myofibroblast    differentiation of rabbit keratocytes requires synergistic TGFβ,    PDGF and integrin signaling. Experimental Eye Research 75(6),    645-657.-   Jhanji, V., Sharma, N., & Vajpayee, R. B. (2011). Management of    keratoconus: current scenario. British Journal of Ophthalmology,    95(8), 1044-1050.-   Jinabhai, A., H. Radhakrishnan, C. O'Donnell. (2010). Pellucid    corneal marginal degeneration: a review. Contact Lens & Anterior    Eye, 34(2), 56-63.-   Johnstone, B., Hering, T. M., Caplan, A. I., Goldberg, V. M., &    Yoo, J. U. (1998). In Vitro Chondrogenesis of Bone Marrow-Derived    Mesenchymal Progenitor Cells. Experimental Cell Research, 238(1),    265-272.-   Kadler, K. E., Baldock, C., Bella, J., & Boot-Handford, R. P.    (2007). Collagens at a glance. Journal of Cell Science, 120(12),    1955-1958.-   Karamichos, D., Hutcheon, A., & Zieske, J. (2011). Transforming    growth factor-β3 regulates assembly of a non-fibrotic matrix in a 3D    corneal model. Journal of Tissue Engineering and Regenerative    Medicine, 5(8), e228-e238.-   Karamichos, D., Zareian, R., Guo, X., Hutcheon, A. E. K.,    Ruberti, J. W., & Zieske, J. D. (2012). Novel in vitro model for    keratoconus disease. Journal of Functional Biomaterials, 3(4),    760-775.-   Kato, Y. and D. Gospodarowicz (1985). Stimulation by glucocorticoid    of the synthesis of cartilage-matrix proteoglycans produced by    rabbit costal chondrocytes in vitro. Journal of Biological    Chemistry, 260(4), 2364-2373.-   C. Kenney, M., & Brown, D. J. (2003). The cascade hypothesis of    keratoconus. Contact Lens and Anterior Eye, 26(3), 139-146.-   Klintworth, G. K. (1999). Advances in the molecular genetics of    corneal dystrophies. American Journal of Ophthalmology, 128(6),    747-754.-   Klintworth, G. K., & Damms, T. (1995). Corneal dystrophies and    keratoconus. Current Opinion in Ophthalmology, 6(4), 44-56.-   Kolambkar, Y. M., Peister, A., Soker, S., Atala, A., &    Guldberg, R. E. (2007). Chondrogenic differentiation of amniotic    fluid-derived stem cells. Journal of Molecular Histology, 38(5),    405-413.-   Krachmer, J. H., Feder, R. S., & Belin, M. W. (1984). Keratoconus    and related noninflammatory corneal thinning disorders. Survey of    Ophthalmology, 28(4), 293-322.-   Ku, J. Y., Niederer, R. L., Patel, D. V., Sherwin, T., &    McGhee, C. N. (2008). Laser scanning in vivo confocal analysis of    keratocyte density in keratoconus. Ophthalmology, 115(5), 845-850.-   Kulyk, W. M., & Hoffman, L. M. (1996). Ethanol exposure stimulates    cartilage differentiation by embryonic limb mesenchyme cells.    Experimental Cell Research, 223(2), 290-300.-   Lee, K. D., et al. (2004). In vitro hepatic differentiation of human    mesenchymal cells. Hepatology, 40(6), 1275-1284.-   Legeais, J.-M., et al. (2001). Nineteen years of penetrating    keratoplasty in the Hotel-Dieu Hospital in Paris. Cornea, 20(6),    603-606.-   Linsenmayer, T. F., Fitch, J. M., & Birk, D. E. (1990). Heterotypic    collagen fibrils and stabilizing collagens. Annals of the New York    Academy of Sciences, 580(1), 143-160.-   Ludwig, A. (2005). The use of mucoadhesive polymers in ocular drug    delivery. Advanced Drug Delivery Reviews, 57(11), 1595-1639.-   Marshall, G. E., Konstas, A. G., & Lee, W. R. (1993). Collagens in    ocular tissues. The British Journal of Ophthalmology, 77(8), 515.-   Mazzotta, C., Traversi, C., Baiocchi, S., Caporossi, O., Bovone, C.,    Sparano, M. C., Caporossi, A. (2008). Corneal healing after    riboflavin ultraviolet-A collagen cross-linking determined by    confocal laser scanning microscopy in vivo: early and late    modifications. American Journal of Ophthalmology, 146(4), 527-533.    e521.-   Meek, K. M., Tuft, S. J., Huang, Y., Gill, P. S., Hayes, S.,    Newton, R. H., & Bron, A. J. (2005). Changes in collagen orientation    and distribution in keratoconus corneas. Investigative Ophthalmology    & Visual Science, 46(6), 1948-1956.-   Mencucci, R., Marini, M., Paladini, I., Sarchielli, E., Sgambati,    E., Menchini, U., & Vannelli, G. B. (2010). Effects of    riboflavin/UVA corneal cross-linking on keratocytes and collagen    fibres in human cornea. Clinical & Experimental Ophthalmology,    38(1), 49-56.-   Mendler, M., Eich-Bender, S. G., Vaughan, L., Winterhalter, K. H., &    Bruckner, P. (1989). Cartilage contains mixed fibrils of collagen    types II, IX, and XI. The Journal of Cell Biology, 108(1), 191-197.-   Menetrey, J., et al. (2000). Growth factors improve muscle healing    in vivo. Journal of Bone & Joint Surgery, British Volume, 82(1),    131-137.-   Niederer, R. L., Perumal, D., Sherwin, T., & McGhee, C. N. J.    (2008). Laser scanning in vivo confocal microscopy reveals reduced    innervation and reduction in cell density in all layers of the    keratoconic cornea. Investigative Ophthalmology & Visual Science,    49(7), 2964-2970.-   Nirmal, H. B., S. R. Bakliwal, S. P. Pawar (2010). In-situ gel: New    trends in controlled and sustained drug delivery system.    International Journal of PharmTech Research, 2(2), 1398-1408.-   Patel, H. Y. et al. (2005). The New Zealand National Eye Bank study    1991-2003: a review of the source and management of corneal tissue.    Cornea, 24(5), 576-582.-   Patel, D., C. McGhee (2013). Understanding keratoconus: what have we    learned from the New Zealand perspective? Clinical and Experimental    Optometry, 96(2), 183-187.-   Peran, M., et al. (2011). Transdifferentiation: why and how? Cell    Biology International, 35(4), 373-379.-   Pramanik, S., Musch, D. C., Sutphin, J. E., & Farjo, A. A. (2006).    Extended long-term outcomes of penetrating keratoplasty for    keratoconus. Ophthalmology, 113(9), 1633-1638.-   Premaraj et al. (2006). Sustained delivery of bioactive cytokine    using a dense collagen gel vehicle collagen gel delivery of    bioactive cytokine. Arch Oral Biol. 51(4), 325-33.-   Rabinowitz, Y. S. (1998). Keratoconus. Survey of Ophthalmology,    42(4), 297-319.-   Rabonitz, Y. S. (2004). Ectatic Disorders of the Cornea. In: The    Cornea, 4th edition. Lippincott Williams & Wilkins.-   Romero-Jiménez, M., Santodomingo-Rubido, J., & Wolffsohn, J. S.    (2010). Keratoconus: a review. Contact Lens and Anterior Eye, 33(4),    157-166.-   Rupenthal, I. D., Green, C. R., & Alany, R. G. (2011). Comparison of    ion-activated in situ gelling systems for ocular drug delivery. Part    2: Precorneal retention and in vivo pharmacodynamic study.    International Journal of Pharmaceutics.-   Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A., &    Benvenisty, N. (2000). Effects of eight growth factors on the    differentiation of cells derived from human embryonic stem cells.    Proceedings of the National Academy of Sciences, 97(21),    11307-11312.-   Shah, M., Foreman, D. M., & Ferguson, M. W. (1995). Neutralisation    of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to    cutaneous rat wounds reduces scarring. Journal of Cell Science,    108(3), 985-1002.-   Sherwin, T., & Brookes, N. H. (2004). Morphological changes in    keratoconus: pathology or pathogenesis. Clinical & Experimental    Ophthalmology, 32(2), 211-217.-   Spoerl, E., Huhle, M., & Seiler, T. (1998). Induction of cross-links    in corneal tissue. Experimental Eye Research, 66(1), 97-103.-   Takahashi, K., S. Yamanaka. (2006). Induction of pluripotent stem    cells from mouse embryonic and adult fibroblast cultures by defined    factors. Cell, 126(4), 663-676.-   Tsang et al. (1995). Characterization of recombinant soluble human    transforming growth factor-beta receptor type II (rhTGF-beta sRII).    Cytokine, 7(5), 389-97.-   Wells, S. M. (2003). Mechanical design of elastic biopolymers.    Physics in Canada, 59(2), 67-74.-   Wernig, M. et al. (2007). In vitro reprogramming of fibroblasts into    a pluripotent ES-cell-like state. Nature, 448(7151), 318-324.-   Willshaw H. et al. (2000). A Handbook of Paediatric Ophthalmology.    Pensord Press: United Kingdom.-   Wilson, S. E., Netto, M., & Ambrosio, R. (2003). Corneal cells:    chatty in development, homeostasis, wound healing, and disease.    American Journal of Ophthalmology, 136(3), 530-536.-   Winter, A., Breit, S., Parsch, D., Benz, K., Steck, E., Hauner, H.,    Richter, W. (2003). Cartilage-like gene expression in differentiated    human stem cell spheroids: A comparison of bone marrow-derived and    adipose tissue-derived stromal cells. Arthritis & Rheumatism, 48(2),    418-429.-   Wollensak, G., Spoerl, E., & Seiler, T. (2003).    Riboflavin/ultraviolet-A-induced collagen crosslinking for the    treatment of keratoconus. American Journal of Ophthalmology, 135(5),    620-627.-   Wollensak, J., & Buddecke, E. (1990). Biochemical studies on human    corneal proteoglycans—a comparison of normal and keratoconic eyes.    Graefe's Archive for Clinical and Experimental Ophthalmology,    228(6), 517-523.-   Worster, A. A., Nixon, A. J., Brower-Toland, B. D., & Williams, J.    (2000). Effect of transforming growth factor β1 on chondrogenic    differentiation of cultured equine mesenchymal stem cells. American    Journal of Veterinary Research, 61(9), 1003-1010.-   Yamanaka, S., H. M. Blau. (2010). Nuclear reprogramming to a    pluripotent state by three approaches. Nature, 465(7299), 704-712.-   Yoon Y. M., Oh C. D., Kim D. Y., Lee Y S, Park J. W., Huh T. L.,    Kang S.

S., Chun J. S. (2000). Epidermal growth factor negatively regulateschondrogenesis of mesenchymal cells by modulating the protein kinaseC-alpha, Erk-1, and p38 MAPK signaling pathways. Biol Chem.275(16):12353-9.

A person of ordinary skill in the art will readily appreciate from thedisclosure that later modifications, substitutions, and/or variationsperforming substantially the same function or achieving substantiallythe same result as embodiments or aspects described herein may beutilised according to such related embodiments or aspects of the presentinvention. Thus, the invention is intended to encompass, within itsscope, the modifications, substitutions, and variations to processes,manufactures, compositions of matter, compounds, means, methods, and/orsteps disclosed herein.

All references, including patents and patent applications, cited in thisspecification are hereby incorporated by reference. No admission is madethat any reference constitutes prior art. Nor does discussion of anyreference constitute an admission that such reference forms part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

1.-46. (canceled)
 47. A method of: (i) treating or preventing acondition associated with a thinning or irregularity of a cornea; (ii)treating or preventing damage or injury of a cornea; or (iii) treatingor preventing a refractive error of the eye, the method comprising:contacting the cornea or the eye with a composition comprising a TGFβ3polypeptide or a variant or fragment thereof, and dexamethasone orderivative thereof or related steroidal agent, thereby treating orpreventing the condition, damage, injury, or refractive error.
 48. Themethod of claim 47, wherein: (a) the TGFβ3 polypeptide consists of theamino acid sequence of SEQ ID NO:1; and/or (b) the dexamethasone isdexamethasone phosphate.
 49. The method of claim 47, wherein: (a) thecomposition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide; and/or(b) the composition comprises 40 to 4000 ng/ml dexamethasone.
 50. Themethod of claim 47, wherein: (a) the composition is administered oncedaily or twice daily; (b) the composition is co-administered with one ormore additional agents for the eye; and/or (c) the composition isadministered in conjunction with use of a contact lens, corneal insert,corneal implant, or intrastromal ring.
 51. The method of claim 50,wherein: (a) the one or more additional agents for the eye are selectedfrom the group consisting of: anaesthetic agents, anti-inflammatoryagents, anti-microbial agents, and lubricants; (b) the contact lens,corneal insert, corneal implant, or intrastromal ring is adapted formoulding or holding corneal shape during and/or following treatment withthe composition; and/or (c) the contact lens, corneal insert, cornealimplant, or intrastromal ring is adapted to act as a carrier for thecomposition or as a composition eluting device.
 52. The method of claim47, wherein: (a) for (i) or (ii), the composition is administered priorto and/or subsequent to corneal collagen crosslinking; or (b) for (ii)or (iii), the method is performed preceding or following refractivesurgery.
 53. The method of claim 47, wherein: (a) the condition isselected from the group consisting of: keratoconus, myopia, andastigmatism; (b) the damage or injury of the cornea is associated withone or more of: an abrasion, tear, ulcer, burn, puncture, or surgery; or(c) the refractive error of the eye is associated with one or more of:myopia, hyperopia, astigmatism, and presbyopia.
 54. A kit comprising:(i) a composition comprising a TGFβ3 polypeptide or a variant orfragment thereof, and dexamethasone or derivative thereof or relatedsteroidal agent; and (ii) one or more contact lenses.
 55. The kit ofclaim 54, wherein: (a) the contact lens, corneal insert, cornealimplant, or intrastromal ring is adapted for moulding or holding cornealshape during and/or following treatment with the composition; and/or (b)the contact lens, corneal insert, corneal implant, or intrastromal ringact as a carrier for the composition or as a composition eluting device.56. The kit of claim 54, wherein: (a) the TGFβ3 polypeptide consists ofthe amino acid sequence of SEQ ID NO:1; and/or (b) the dexamethasone isdexamethasone phosphate.
 57. The kit of claim 54, wherein thecomposition comprises one or more of: (a) 10 to 100 ng/ml of the TGFβ3polypeptide; (b) 40 to 4000 ng/ml dexamethasone; (c) a formulation foradministration once daily or twice daily; or (d) a co-formulation withone or more additional agents for the eye.
 58. The kit of claim 54,wherein: (a) the kit includes one or more additional agents for the eye;(b) the kit includes a contact lens solution; and/or (c) the kitincludes instructions for use.
 59. The kit of claim 58, wherein the oneor more additional agents for the eye are selected from the groupconsisting of: anaesthetic agents, anti-inflammatory agents,anti-microbial agents, and lubricants.
 60. The kit of claim 54, whereinthe kit is used for: (i) the treatment or prevention of a refractiveerror of the eye; (ii) the treatment or prevention of a cornealcondition selected from the group consisting of: keratoconus, myopia,hyperopia, astigmatism, presbyopia, and stromal dystrophies; or (iii)the treatment of a corneal condition selected from the group consistingof: an abrasion, tear, ulcer, burn, puncture, corneal melt, and surgicalinjury.
 61. A method of inducing collagen type II expression in akeratocyte, comprising: contacting the keratocyte with a compositioncomprising a TGFβ3 polypeptide or a variant or fragment thereof, anddexamethasone or derivative thereof or related steroidal agent, therebyinducing collagen type II expression in the keratocyte.
 62. The methodof claim 61, wherein: (a) the TGFβ3 polypeptide consists of the aminoacid sequence of SEQ ID NO:1; and/or (b) the dexamethasone isdexamethasone phosphate.
 63. The method of claim 61, wherein: (a) thecomposition comprises 10 to 100 ng/ml of the TGFβ3 polypeptide; and/or(b) the composition comprises 40 to 4000 ng/ml dexamethasone.
 64. Themethod of claim 61, wherein: (a) the composition is formulated foradministration via a contact lens, a corneal insert, a corneal implant,or an intrastromal ring; (b) the composition is formulated foradministration as a solution, gel, cream, or emulsion; and/or (c) thecomposition is formulated for administration in vivo or ex vivo.
 65. Amethod of: (i) treating or preventing a condition associated with athinning or irregularity of a cornea; (ii) treating or preventing damageor injury of a cornea; or (iii) treating or preventing a refractiveerror of the eye, the method comprising: co-administering to the corneaor to the eye a composition comprising a TGFβ3 polypeptide or a variantor fragment thereof, and a composition comprising dexamethasone orderivative thereof or related steroidal agent, thereby treating orpreventing the condition, damage, injury, or refractive error.
 66. Amethod of inducing collagen type II expression in a keratocyte,comprising: co-administering to the keratocyte a composition comprisinga TGFβ3 polypeptide or a variant or fragment thereof, and a compositioncomprising dexamethasone or derivative thereof or related steroidalagent, thereby inducing collagen type II expression in the keratocyte.